Nucleic acid encoding retinoic acid receptor

ABSTRACT

The present invention provides amino acid sequences of peptides that are encoded by genes within the human genome, the nuclear hormone receptor peptides of the present invention. The present invention specifically provides isolated peptide and nucleic acid molecules, methods of identifying orthologs and paralogs of the nuclear hormone receptor peptides, and methods of identifying modulators of the nuclear hormone receptor peptides.

RELATED APPLICATIONS

The present application claims priority to provisional application U.S.Ser. No. 60/239,117, filed Oct. 11, 2000.

FIELD OF THE INVENTION

The present invention is in the field of nuclear hormone receptorproteins that are related to the retinoic acid receptor nuclear hormonereceptor subfamily, recombinant DNA molecules, and protein production.The present invention specifically provides novel nuclear hormonereceptor peptides and proteins and nucleic acid molecules encoding suchpeptide and protein molecules, all of which are useful in thedevelopment of human therapeutics and diagnostic compositions andmethods.

BACKGROUND OF THE INVENTION

Nuclear Hormone Receptors

Cellular signal transduction is a fundamental mechanism whereby externalstimuli that regulate diverse cellular processes are relayed to theinterior of cells. The biochemical pathways through which signals aretransmitted within cells comprise a circuitry of directly orfunctionally connected interactive proteins. One of the key biochemicalmechanisms of signal transduction involves the activation of genetranscription. Many of the transcription activation pathways arecontrolled by the action of intracellular receptors (IRs), such asmembers of the nuclear hormone receptor family of proteins and theirligands. The binding of a ligand to a nuclear hormone receptor serves totranslate signals generated from a variety of cellular events.

Intracellular receptors (IRs) form a class of structurally relatedgenetic regulators scientists have named “ligand dependent transcriptionfactors.” R. M. Evans, 240 Science, 889 (1988). Nuclear hormonereceptors are a recognized subset of the IRs, including the progesteronereceptor (PR), androgen receptor (AR), estrogen receptor (ER),glucocorticoid receptor (GR) and mineralocorticoid receptor (MR).Regulation of a gene by such factors requires both the IR itself and acorresponding ligand which has the ability to selectively bind to the IRin a way that alters gene transcription.

Ligands to the IRs can include low molecular weight native molecules,such as the hormones progesterone, estrogen and testosterone, as well assynthetic derivative compounds such as medroxyprogesterone acetate,diethylstilbesterol and 19-nortestosterone. These ligands, when presentin the fluid surrounding a cell, pass through the outer cell membrane bypassive diffusion and bind to specific IR proteins to create aligand/receptor complex. This complex then translocates to the cell'snucleus, where it binds to a specific gene or genes present in thecell's DNA. Once bound to DNA, the complex modulates the production ofthe protein encoded by that gene. In this regard, a compound which bindsand IR and mimics the effect of the native ligand is referred to as an“agonist”, while a compound that inhibits the effect of the nativeligand is called an “antagonist.”

Ligands to the nuclear hormone receptors are known to play an importantrole in health of both women and men. For example, the native femaleligand, progesterone, as well as synthetic analogues, such as norgestrel(18-homonorethisterone) and norethisterone(17.alpha.-ethinyl-19-nortestosterone), are used in birth controlformulations, typically in combination with the female hormone estrogenor synthetic estrogen analogues, as effective modulators of both PR andER. On the other hand, antagonists to PR are potentially useful intreating chronic disorders, such as certain hormone dependent cancers ofthe breast, ovaries, and uterus, and in treating non-malignantconditions such as uterine fibroids and endometriosis, a leading causeof infertility in women. Similarly, AR antagonists, such as cyproteroneacetate and flutamide have proved useful in the treatment of hyperplasiaand cancer of the prostate.

Nuclear hormone hormones, one sub-family of IRs, are potent modulatorsof transcriptional events that together regulate the complex processesassociated with differentiation homeostasis and development. Themechanism of action of these molecules is related in that the effectormolecule binds to a specific intracellular receptor. This binding altersthe structure of the receptor, thus increasing its affinity for specificrecognition sites within the regulatory region of target genes. In thisway, the nuclear hormone directs a program of events that leads to achange in cell phenotype.

Nuclear hormone hormones, thyroid hormones and certain vitamins canregulate cellular differentiation morphogenesis and homeostasis bybinding to specific intracellular receptor proteins. Ligand activatedreceptor complexes are capable of activating or repressing transcriptionof a specific set of target genes. Thus, the receptor proteins arecapable of reprogramming cellular function at the genomic level inresponse to hormonal or vitamin signals.

Retinoic Acids and Retinoic Acid Receptors

The protein provided by the present invention is a novel retinoic acidreceptor isoform; the new isoform provided herein is an ortholog of therat retinoic acid receptor alpha 2. Consequently, the new retinoic acidreceptor isoform provided by the present invention may be named humanretinoic acid receptor alpha 2. As used herein, the term retinoic acid(RA) is synonymous with retinoid, and the term retinoic acid receptor issynonymous with retinoid receptor.

Retinoids, or vitamin A metabolites/derivatives, have been determined toplay essential roles in many aspects of development, metabolism andreproduction in vertebrates (see, for example, The Retinoids, SecondEdition, Sporn et al. (Raven Press, New York, 1994)). There are twoclasses of retinoid receptors: the retinoic acid receptors (RARs), whichbind to both all-trans retinoic acid (atRA) and 9-cis retinoic acid(9cRA), and the retinoid X receptors (RXRs), which bind only to 9cRA.These receptors modulate ligand-dependent gene expression by interactingas RXR/RAR heterodimers or RXR homodimers on specific target gene DNAsequences known as hormone response elements. In addition to their rolein retinoid signalling, RXRs also serve as heterodimeric partners ofnuclear receptors for vitamin D, thyroid hormone, and peroxisomeproliferators (reviewed by Mangelsdorf et al., at pages 319-349 of TheRetinoids, Second Edition, Spom et al. (Raven Press, New York, 1994)).

A number of studies have demonstrated that retinoids are essential fornormal growth, vision, tissue homeostasis, reproduction and overallsurvival (for reviews and references, See Sporn et al., The Retinoids,Vols. 1 and 2, Sporn et al., eds., Academic Press, Orlando, Fla.(1984)). For example, retinoids have been shown to be vital to themaintenance of skin homeostasis and barrier function in mammals (Fisher,G. J., and Voorhees, J. J., FASEB J. 10:1002-1013 (1996)). Retinoids arealso apparently crucial during embryogenesis, since offspring of damswith vitamin A deficiency (VAD) exhibit a number of developmentaldefects (Wilson, J. G., et al., Am. J. Anat. 92:189-217 (1953);Morriss-Kay, G. M., and Sokolova, N., FASEB J. 10:961-968 (1996)). Withthe exceptions of those on vision (Wald, G., et al., Science 162:230-239(1968)) and spermatogenesis in mammals (van Pelt, H. M. M., and DeRooij, D. G., Endocrinology 128:697-704 (1991)), most of the effectsgenerated by VAD in animals and their fetuses can be prevented and/orreversed by retinoic acid (RA) administration (Wilson, J. G., et al.,Am. J. Anat. 92:189-217 (1953); Thompson et al., Proc. Royal Soc.159:510-535 (1964); Morriss-Kay, G. M., and Sokolova, N., FASEB J.10:961-968 (1996)). The dramatic teratogenic effects of maternal RAadministration on mammalian embryos (Shenefelt, R. E., Teratology 5,103-108 (1972); Kessel, M., Development 115:487-501 (1992); CreechKraft, J., In Retinoids in Normal Development and Teratogenesis, G. M.Morriss-Kay, ed., Oxford University Press, Oxford, UK, pp. 267-280(1992)), and the marked effects of topical administration of retinoidson embryonic development of vertebrates and limb regeneration inamphibians (Mohanty-Hejmadi et al., Nature 355:352-353 (1992); Tabin, C.J., Cell 66:199-217 (1991)), have contributed to the notion that RA mayhave critical roles in morphogenesis and organogenesis.

Many synthetic structural analogues of all-trans retinoic acid or9-cis-retinoic acid, commonly termed “retinoids”, have been described inthe literature to date. Some of these molecules are able to bind to, andspecifically activate, the RARs or, on the other hand, the RXRs.Furthermore, some analogues are able to bind to, and activate aparticular RAR receptor subtype (.alpha., .beta. or .gamma.). Finally,other analogues do not exhibit any particular selective activity withregard to these different receptors. In this respect, and by way ofexample, 9-cis-retinoic acid activates the RARs and the RXRs at one andthe same time without any noteworthy selectivity for either of thesereceptors (nonspecific agonist ligand), whereas all-trans retinoic acidselectively activates the RARs (RAR-specific agonist ligand), with allsubtypes being included. In a general manner, and qualitatively, a givensubstance (or ligand) is said to be specific for a given family ofreceptors (or, respectively, for a particular receptor of this family)when the said substance exhibits an affinity for all the receptors ofthis family (or, respectively, for the particular receptor of thisfamily) which is stronger than that which it otherwise exhibits for allthe receptors of any other family (or, respectively, for all the otherreceptors, of this same family or not).

The genetic activities of the RA signal are mediated through the twofamilies of receptors—the RAR family and the RXR family—which belong tothe superfamily of ligand-inducible transcriptional regulatory factorsthat include steroid/thyroid hormone and vitamin D3 receptors (forreviews see Leid et al., TIBS 17:427-433 (1992); Chambon, P., Semin.Cell Biol. 5:115-125 (1994); Chambon, P., FASEB J. 10:940-954 (1996);Giguere, V., Endocrinol. Rev. 15:61-79 (1994); Mangelsdorf, D. J., andEvans, R. M., Cell 83:841-850 (1995); Gronemeyer, H., and Laudet, V.,Protein Profile 2:1173-1236 (1995)).

RARs are the critical factors in tissue differentiation and developmentThey are up-regulated in rapidly dividing cells and tumors. RARs play animportant role in lymphocyte activation. Synthetic antagonists ofretinoic acid receptors can inhibit delayed type hypersensitivity (DTH).Growth factors and carotene regulate RXR expression levels. For example,granulocyte macrophage colony-stimulating factor induces retinoic acidreceptors in myeloid leukemia cells.

Retinoic acid receptors can form heterodimers with other nuclearreceptors. The protein provided by the present invention can be used asa probe to detect possible interactions in the two-hybrid assay.Synthetic peptides that mimic dimerization surface can disruptintermolecular interactions between these receptors. RAR generearrangements are the primary causes of some types of leukemia andprovide a convenient genetic marker for malignant cell lines. A numberof retinoic acid derivatives are used in treatment of myelodysplasticdisorders. They are designed to bind and activate RXRs. Beta-cotene canprevent skin tumor formation in mouse models.N-(4-hydroxyphenyl)retinarmide can delay onset of dysplasia in bronchi.Different chemopreventive drugs can be designed to target individualretinoic receptors. The sequences provided by the present invention maybe used to design high affinity chemopreventive compounds.

Although both the RARs and RXRs respond to all-trans-retinoic acid invivo, the receptors differ in several important aspects. First, the RARsand RXRs are significantly divergent in primary structure (e.g., theligand binding domains of RAR.alpha and RXR.alpha. have only 27% aminoacid identity). These structural differences are reflected in thedifferent relative degrees of responsiveness of RARs and RXRs to variousvitamin A metabolites and synthetic retinoids. In addition, distinctlydifferent patterns of tissue distribution are seen for RARs and RXRs.For example, in contrast to the RARS, which are generally not expressedat high levels in the visceral tissues, RXR.alpha. mRNA has been shownto be most abundant in the liver, kidney, lung, muscle and intestine.Finally, the RARs and RXRs have different target gene specificity. Forexample, response elements have recently been identified in the cellularretinal binding protein type II (CRBPII) and apolipoprotein A1 geneswhich confer responsiveness to RXR, but not RAR. Furthermore, RAR hasalso been recently shown to repress RXR-mediated activation through theCRBPII RXR response element (Manglesdorf et al., Cell, 66:555-61(1991)). These data indicate that two retinoic acid responsive pathwaysare not simply redundant, but instead manifest a complex interplay.Recently, Heyman et al. (Cell, 68:397-406 (1992)) and Levin et al.(Nature, 355:359-61 (1992)) independently demonstrated that9-cis-retinoic acid is a natural endogenous ligand for the RXRs.9-cis-retinoic acid was shown to bind and transactivate the RXRs, aswell as the RARs, and therefore appears to act as a “bifunctional”ligand.

RAR Receptors

Receptors belonging to the RAR family (RAR.alpha., .beta. and .gamma.and their isoforms) are activated by both all-trans- and 9-cis-RA (Leidet al., TIBS 17:427-433 (1992); Chambon, P., Semin. Cell Biol. 5:115-125(1994); Dolle, P., et al., Mech. Dev. 45:91-104 (1994); Chambon, P.,FASEB J. 10:940-954 (1996)). Within a given species, the DNA binding (C)and the ligand binding (E) domains of the three RAR types are highlysimilar, whereas the C-terminal domain F and the middle domain D exhibitno or little similarity. The amino acid sequences of the three RAR typesare also notably different in their B regions, and their main isoforms(.alpha.1 and .alpha.2, .beta.1 to .beta.4, and .gamma.1 and .gamma.2)further differ in their N-terminal A regions (Leid et al., TIBS17:427-433 (1992)). Amino acid sequence comparisons have revealed thatthe interspecies conservation of a given RAR type is greater than thesimilarity found between the three RAR types within a given species(Leid et al., TIBS 17:427-433 (1992)). This interspecies conservation isparticularly striking in the N-terminal A regions of the variousRAR.alpha., .beta. and .gamma. isoforms, whose A region amino acidsequences are quite divergent. Taken together with the distinctspatio-temporal expression patterns observed for the transcripts of eachRAR and RXR type in the developing embryo and in various adult mousetissues (Zelent, A., et al., Nature 339:714-717 (1989); Dolle, P., etal., Nature 342:702-705 (1989); Dolleet al., Development 110:1133-1151(1990); Ruberte et al., Development 108:213-222 (1990); Ruberte et al.,Development 111:45-60 (1991); Mangelsdorf et al., Genes & Dev. 6:329-344(1992)), this interspecies conservation has suggested that each RAR type(and isoform) may perform unique functions. This hypothesis is furthersupported by the finding that the various RAR isoforms contain twotranscriptional activation functions (AFs) located in the N-terminal A/Bregion (AF-1) and in the C-terminal E region (AF-2), which cansynergistically, and to some extent differentially, activate variousRA-responsive promoters (Leid et al., TIBS 17:427433 (1992); Nagpal, S.,et al., Cell 70:1007-1019 (1992); Nagpal, S., et al., EMBO J.12:2349-2360 (1993)).

RXR Receptors

Unlike the RARs, members of the retinoid X receptor family (RXR.alpha.,.beta. and .gamma.) are activated exclusively by 9-cis-RA (Chambon, P.,FASEB J. 10:940-954 (1996); Chambon, P., Semin. Cell Biol. 5:115-125(1994); Dolle, P., et al., Mech. Dev. 45:91-104 (1994); Linney, E.,Current Topics in Dev. Biol. 27:309-350 (1992); Leid et al., TIBS17:427-433 (1992); Kastner et al., in Vitamin A in Health and Disease,R. Blomhoff, ed., Marcel Dekker, New York (1993)). However, the RXRscharacterized to date are similar to the RARs in that the different RXRtypes also differ markedly in their N-terminal A/B regions (Leid et al.,TIBS 17:427-433 (1992); Leid et al., Cell 68:377-395 (1992); Mangelsdorfet al., Genes and Dev. 6:329-344 (1992)), and contain the sametranscriptional activation functions in their N-terminal A/B region andC-terminal E region (Leid et al., TIBS 17:427-433 (1992); Nagpal, S., etal., Cell 70:1007-1019 (1992); Nagpal, S., et al., EMBO J.12:2349-2360(1993)).

RXR.alpha. and RXR.beta. have a widespread (possibly ubiquitous)expression pattern during mouse development and in the adult animal,being found in all fetal and adult tissues thus far examined(Mangelsdorf, D. J., et al., Genes & Devel. 6:329-344 (1992); Dolle, P.,et al., Mech. Devel. 45:91-104 (1994); Nagata, T., et al., Gene142:183-189 (1994)). RXR.gamma. transcripts, however, appear to have amore restricted distribution, being expressed in developing skeletalmuscle in the embryo (where their expression persists throughout life),in the heart (after birth), in sensory epithelia of the visual andauditory systems, in specific structures of the central nervous system,and in tissues involved in thyroid hormone homeostasis, e.g., thethyroid gland and thyrotrope cells in the pituitary (Mangelsdorf, D. J.,et al., Genes & Devel. 6:329-344 (1992); Dolle, P., et al., Mech. Devel.45:91-104 (1994); Sugawara, A., et al., Endocrinology 136:1766-1774(1995); Liu, Q., and Linney, E., Mol. Endocrinol. 7:651-658 (1993)).

It is currently unclear whether all the molecular properties of RXRscharacterized in vitro are relevant for their physiological functions invivo. In particular, it is unknown under what conditions these receptorsact as 9-cis-RA-dependent transcriptional regulators (Chambon, P.,Semin. Cell Biol. 5:115-125 (1994)). The knock-outs of RXR.alpha. andRXR.beta. in the mouse have provided some insight into the physiologicalfunctions of these receptors. For example, the ocular and cardiacmalformations observed in RXR.alpha . . . sup.−/− fetuses (Kastner, P.,et al., Cell 78:987-1003 (1994); Sucov, H. M., et al., Genes & Devel.8:1007-1018 (1994)) are similar to those found in the fetal VADsyndrome, thus suggesting an important function of RXR.alpha. in thetransduction of a retinoid signal during development. The involvement ofRXRs in retinoid signaling is further supported by studies of compoundRXR.alpha./RAR mutants, which reveal defects that are either absent orless severe in the single mutants (Kastner, P., et al., Cell 78:987-1003(1994); Kastner, P., et al., Cell 83:859-869 (1995)). Interestingly,however, knockout of RXR.gamma. in the mouse induces no overtdeleterious effects, and RXR.gamma . . . sup.−/− homozygotes which arealso RXR.alpha . . . sup.−/− or RXR.beta . . . sup.−/− exhibit noadditional abnormalities beyond those seen in RXR.alpha . . . sup.−/−,RXR.beta . . . sup.−/− and fetal VAD syndrome fetuses (Krezel, W., etal., Proc. Natl. Acad. Sci. USA 93(17):9010-9014 (1996)), suggestingthat RXR.gamma., despite its highly tissue-specific expression patternin the developing embryo, is dispensable for embryonic development andpostnatal life in the mouse. The observation that live-born RXR.gamma .. . sup.−/−/RXR.beta . . . sup.−/−/RXR.alpha . . . sup.−/− mutants cangrow to reach adult age (Krezel et al., Proc. Natl. Acad. Sci. USA93(17):9010-9014 (1996)) indicates that a single RXR.alpha. allele issufficient to carry out all of the vital developmental and postnatalfunctions of the RXR family of receptors, particularly all of thedevelopmental functions which depend on RARs and may require RXRpartnership (Dolle, P., et al., Mech. Dev. 45:91-104 (1994); Kastner,P., et al., Cell 83:859-869 (1995)). Furthermore, the finding thatRXR.alpha . . . sup.−/−/RXR.gamma . . . sup.−/− double mutant embryosare not more affected than are single RXR.alpha . . . sup.−/− mutants(Krezel et al., Proc. Natl. Acad. Sci. USA 93(17):9010-9014 (1996))clearly shows that RXR.beta.alone can also perform some of thesefunctions. Therefore, the fact that RXR.alpha. alone and, to a certainextent RXR.beta. alone, are sufficient for the completion of a number ofdevelopmental RXR functions, clearly indicates the existence of a largedegree of functional redundancy amongst RXRs. In this respect, the RXRsituation is different from that of RARs, since all of types of RARdouble mutants displayed much broader sets of defects than singlemutants Rowe, A., et al., Develop. 111:771-778 (1991); Lohnes, D., etal., Develop. 120:2723-2748 (1994); Mendelsohn, C., Develop.120:2749-2771 (1994)).

Retinoid Binding to RAR and RXR Receptors

The crystal structures of the ligand-binding domains (LBDs) of the RARsand RXRs have recently been elucidated (Bourget, W., et al., Nature375:377-382 (1995); Renaud, J. P., et al., Nature 378:681-689 (1995);Wurtz, J. M., et al., Nature Struct. Biol. 3:87-94 (1996)). Among thevarious RAR types, substantial amino acid sequence identity is observedin these domains: comparison of the LBDs of RAR.alpha., RAR.beta. andRAR.gamma. indicates that only three amino acid residues are variable inthe ligand-binding pocket of these receptors. These residues apparentlyaccount for the fact that the various RAR types exhibit some selectivityin binding certain synthetic retinoids (Chen, J.-Y., et al., EMBO J.14(6):1187-1197 (1995); Renaud, J. P., et al., Nature 378:681-689(1995)), and consideration of these divergent residues can be used todesign RAR type-specific synthetic retinoids which may be agonistic orantagonistic (Chambon, P., FASEB J. 10:940-954 (1996)). This designapproach may be extendable generally to other nuclear receptors, such asthyroid receptor .alpha. (Wagner, R. L., et al., Nature 378:690-697(1995)), the ligand-binding pockets of which may chemically andstructurally resemble those of the RARs (Chambon, P., FASEB J.10:940-954 (1996)). Conversely, molecular modeling of the ligand-bindingpocket of the RXRs demonstrates that there are no overt differences inamino acid composition between RXR.alpha., RXR.beta. and RXR.gamma.(Bourguet, W., et al., Nature 375:377-382 (1995); Wurtz, J. M., et al.,Nature Struct. Biol. 3:87-94 (1996)), suggesting that design oftype-specific synthetic ligands for the RXRs may be more difficult thanfor the RARs (Chambon, P., FASEB J. 10:940-954 (1996)).

Retinoid Signaling Through RAR:RXR Heterodimers

Nuclear receptors (NRs) are members of a superfamily of ligand-inducibletranscriptional regulatory factors that include receptors for steroidhormones, thyroid hormones, vitamin D3 and retinoids (Leid, M., et al.,Trends Biochem. Sci. 17:427433 (1992); Leid, M., et al., Cell 68:377-395(1992); and Linney, E. Curr. Top. Dev. Biol., 27:309-350 (1992)). NRsexhibit a modular structure which reflects the existence of severalautonomous functional domains. Based on amino acid sequence similaritybetween the chicken estrogen receptor, the human estrogen andglucocorticoid receptors, and the v-erb-A oncogene (Krust, A., et al.,EMBO J. 5:891-897 (1986)), defined six regions—A, B, C, D, E and F—whichdisplay different degrees of evolutionary conservation amongst variousmembers of the nuclear receptor superfamily. The highly conserved regionC contains two zinc fingers and corresponds to the core of theDNA-binding domain (DBD), which is responsible for specific recognitionof the cognate response elements. Region E is functionally complex,since in addition to the ligand-binding domain (LBD), it contains aligand-dependent activation function (AF-2) and a dimerizationinterface. An autonomous transcriptional activation function (AF-1) ispresent in the non-conserved N-terminal A/B regions of the steroidreceptors. Interestingly, both AF-1 and AF-2 of steroid receptorsexhibit differential transcriptional activation properties which appearto be both cell type and promoter context specific (Gronemeyer, H. Annu.Rev. Genet. 25:89-123 (1991)).

As described above, the all-trans (T-RA) and 9-cis (9C-RA) retinoic acidsignals are transduced by two families of nuclear receptors, RAR.alpha., .beta. and .gamma. (and their isoforms) are activated by bothT-RA and 9C-RA, whereas RXR .alpha., .beta. and .gamma. are exclusivelyactivated by 9C-RA (Allenby, G. et al., Proc. Natl. Acad. Sci. USA90:30-34 (1993)). The three RAR types differ in their B regions, andtheir main isoforms (.alpha.1 and .alpha.2, .beta. 1-4, and .gamma.1 and.gamma.2) have different N-terminal A regions (Leid, M. et al., TrendsBiochem. Sci. 17:427-433 (1992)). Similarly, the RXR types differ intheir A/B regions (Mangelsdorf, D. J. et al., Genes Dev. 6:329-344(1992)).

The E-region of RARs and RXRs has also been shown to contain adimerization interface (Yu, V. C. et al., Curr. Opin. Biotechnol.3:597-602 (1992)). Most interestingly, it was demonstrated that RAR/RXRheterodimers bind much more efficiently in vitro than homodimers ofeither receptor to a number of RA response elements (RAREs) (Yu, V. C.et al., Cell 67:1251-1266 (1991); Berrodin, T. J. et al., Mol.Endocrinol 6:1468-1478 (1992); Bugge, T. H. et al., EMBO J. 11:1409-1418(1992); Hall, R. K. et al., Mol. Cell. Biol. 12: 5527-5535 (1992);Hallenbeck P. L. et al., Proc. Natl. Acad. Sci. USA 89:5572-5576 (1992);Husmann, M. et al., Biochem. Biophys. Res. Commun. 187:1558-1564 (1992);Kliewer, S. A. et al., Nature 355:446-449 (1992); Leid, M. et al., Cell68:377-395 (1992); Marks, M. S. et al., EMBO J. 11:1419-1435 (1992);Zhang, X. K. et al., Nature 355:441-446 (1992)). RAR and RXRheterodimers are also preferentially formed in solution in vitro (Yu, V.C. et al., Cell 67:1251-1266 (1991); Leid, M. et al., Cell 68:377-395(1992); Marks, M. S. et al., EMBO J. 11:1419-1435 (1992)), although theaddition of 9C-RA appears to enhance the formation of RXR homodimers invitro (Lehman, J. M. et al., Science 258:1944-1946 (1992); Zhang, X. K.et al., Nature 358:587-591 (1992b)).

It has been shown that activation of RA-responsive promoters likelyoccurs through RAR:RXR heterodimers rather than through homodimers (Yu,V. C. et al., Cell 67:1251-1266 (1991); Leid et al., Cell 68:377-395(1992b); Durand et al., Cell 71:73-85 (1992); Nagpal et al., Cell70:1007-1019 (1992); Zhang, X. K., et al., Nature 355, 441-446 (1992);Kliewer et al., Nature 355:446-449 (1992); Bugge et al., EMBO J. 11:1409-1418 (1992); Marks et al., EMBO J. 11:1419-1435 (1992); Yu, V. C.et al., Cur. Op. Biotech. 3:597-602 (1992); Leid et al., TIBS 17:427-433(1992); Laudet and Stehelin, Curr. Biol. 2:293-295 (1992); Green, S.,Nature 361:590-591 (1993)). The RXR portion of these heterodimers hasbeen proposed to be silent in retinoid-induced signaling (Kurokawa, R.,et al., Nature 371:528-531 (1994); Forman, B. M., et al., Cell81:541-550 (1995); Mangelsdorf, D. J., and Evans, R. M., Cell 83:835-850(1995)), although conflicting results have been reported on this issue(Apfel, C. M., et al., J. Biol. Chem. 270(51):30765-30772 (1995); seeChambon, P., FASEB J. 10:940-954 (1996) for review). Although theresults of these studies strongly suggest that RAR/RXR heterodimers areindeed fractional units that transduce the RA signal in vivo, it isunclear whether all of the suggested heterodimeric combinations occur invivo (Chambon, P., Semin. Cell Biol. 5:115-125 (1994)). Thus, the basisfor the highly pleiotropic effect of retinoids may reside, at least inpart, in the control of different subsets of retinoid-responsivepromoters by cell-specifically expressed heterodimeric combinations ofRAR:RXR types (and isoforms), whose activity may be in turn regulated bycell-specific levels of all-trans- and 9-cis-RA (Leid et al., TIBS17:427-433 (1992)).

The RXR receptors may also be involved in RA-independent signaling. Forexample, the observation of aberrant lipid metabolism in the Sertolicells of RXR.beta . . . sup.−/− mutant animals suggests that functionalinteractions may also occur between RXR.beta. and the peroxisomalproliferator-activated receptor signaling pathway (WO 94/26100; Kastner,P., et al., Genes & Devel. 10:80-92 (1996)).

For a further review of retinoic acid receptors, see: Shimizu et al.,Cancer Res 2000 August 15;60(16):4544-9; Ponnamperuma et al., NutrCancer 2000;37(1):82-8; Yoshimura et al., J Med Chem 2000 Jul27;43(15):2929-37; Kurie et al., Clin Cancer Res 2000 Aug;6(8):2973-9;Lee et al., J Biol Chem 2000 Aug 17; and Sainty et al., Blood 2000 Aug15;96(4):1287-96.

The discovery of a new human nuclear hormone receptor proteins and thepolynucleotides encoding it satisfies a need in the art by providing newcompositions which are useful in the diagnosis, prevention and treatmentof biological processes associated with abnormal or unwanted proteingene activation.

SUMMARY OF THE INVENTION

The present invention is based in part on the identification of aminoacid sequences of human nuclear hormone receptor peptides and proteinsthat are related to the retinoic acid receptor nuclear hormone receptorsubfamily, as well as allelic variants and other mammalian orthologsthereof. These unique peptide sequences, and nucleic acid sequences thatencode these peptides, can be used as models for the development ofhuman therapeutic targets, aid in the identification of therapeuticproteins, and serve as targets for the development of human therapeuticagents that modulate nuclear hormone receptor activity in cells andtissues that express the nuclear hormone receptor. Experimental data asprovided in FIG. 1 indicates that nuclear hormone receptors of thepresent invention are expressed in humans in breast, brain, head-necktissue, testis, placenta, retina, liver, kidney, HeLa cell tissue, andfetal liver-spleen.

DESCRIPTION OF THE FIGURE SHEETS

FIG. 1 provides the nucleotide sequence of a cDNA molecule or transcriptsequence that encodes the nuclear hormone receptor protein of thepresent invention. (SEQ ID NO:1) In addition, structure and functionalinformation is provided, such as ATG start, stop and tissuedistribution, where available, that allows one to readily determinespecific uses of inventions based on this molecular sequence.Experimental data as provided in FIG. 1 indicates that nuclear hormonereceptors of the present invention are expressed in humans in breast,brain, head-neck tissue, testis, placenta, retina, liver, kidney, HeLacell tissue, and fetal liver-spleen.

FIG. 2 provides the predicted amino acid sequence of the nuclear hormonereceptor of the present invention. (SEQ ID NO:2) In addition structureand functional information such as protein family, function, andmodification sites is provided where available, allowing one to readilydetermine specific uses of inventions based on this molecular sequence.

FIG. 3 provides genomic sequences that span the gene encoding thenuclear hormone receptor protein of the present invention. (SEQ ID NO:3)In addition structure and functional information, such as intron/exonstructure, promoter location, etc., is provided where available,allowing one to readily determine specific uses of inventions based onthis molecular sequence. As illustrated in FIG. 3, known SNP variationsinclude C4084G, G6482A, C8066G, T8699C, C1289T, and C14442T.

DETAILED DESCRIPTION OF THE INVENTION

General Description

The present invention is based on the sequencing of the human genome.During the sequencing and assembly of the human genome, analysis of thesequence information revealed previously unidentified fragments of thehuman genome that encode peptides that share structural and/or sequencehomology to protein/peptide/domains identified and characterized withinthe art as being a nuclear hormone receptor protein or part of a nuclearhormone receptor protein and are related to the retinoic acid receptornuclear hormone receptor subfamily. Utilizing these sequences,additional genomic sequences were assembled and transcript and/or cDNAsequences were isolated and characterized. Based on this analysis, thepresent invention provides amino acid sequences of human nuclear hormonereceptor peptides and proteins that are related to the retinoic acidreceptor nuclear hormone receptor subfamily, nucleic acid sequences inthe form of transcript sequences, cDNA sequences and/or genomicsequences that encode these nuclear hormone receptor peptides andproteins, nucleic acid variation (allelic information), tissuedistribution of expression, and information about the closest art knownprotein/peptide/domain that has structural or sequence homology to thenuclear hormone receptor of the present invention.

In addition to being previously unknown, the peptides that are providedin the present invention are selected based on their ability to be usedfor the development of commercially important products and services.Specifically, the present peptides are selected based on homology and/orstructural relatedness to known nuclear hormone receptor proteins of theretinoic acid receptor nuclear hormone receptor subfamily and theexpression pattern observed. Experimental data as provided in FIG. 1indicates that nuclear hormone receptors of the present invention areexpressed in humans in breast, brain, head-neck tissue, testis,placenta, retina, liver, kidney, HeLa cell tissue, and fetalliver-spleen. The art has clearly established the commercial importanceof members of this family of proteins and proteins that have expressionpatterns similar to that of the present gene. Some of the more specificfeatures of the peptides of the present invention, and the uses thereof,are described herein, particularly in the Background of the Inventionand in the annotation provided in the Figures, and/or are known withinthe art for each of the known retinoic acid receptor family or subfamilyof nuclear hormone receptor proteins.

Specific Embodiments

Peptide Molecules

The present invention provides nucleic acid sequences that encodeprotein molecules that have been identified as being members of thenuclear hormone receptor family of proteins and are related to theretinoic acid receptor nuclear hormone receptor subfamily (proteinsequences are provided in FIG. 2, transcript/cDNA sequences are providedin FIG. 1 and genomic sequences are provided in FIG. 3). The peptidesequences provided in FIG. 2, as well as the obvious variants describedherein, particularly allelic variants as identified herein and using theinformation in FIG. 3, will be referred herein as the nuclear hormonereceptor peptides of the present invention, nuclear hormone receptorpeptides, or peptides/proteins of the present invention.

The present invention provides isolated peptide and protein moleculesthat consist of, consist essentially of, or comprise the amino acidsequences of the nuclear hormone receptor peptides disclosed in the FIG.2, (encoded by the nucleic acid molecule shown in FIG. 1,transcript/cDNA or FIG. 3, genomic sequence), as well as all obviousvariants of these peptides that are within the art to make and use. Someof these variants are described in detail below.

As used herein, a peptide is said to be “isolated” or “purified” when itis substantially free of cellular material or free of chemicalprecursors or other chemicals. The peptides of the present invention canbe purified to homogeneity or other degrees of purity. The level ofpurification will be based on the intended use. The critical feature isthat the preparation allows for the desired function of the peptide,even if in the presence of considerable amounts of other components (thefeatures of an isolated nucleic acid molecule is discussed below).

In some uses, “substantially free of cellular material” includespreparations of the peptide having less than about 30% (by dry weight)other proteins (i.e., contaminating protein), less than about 20% otherproteins, less than about 10% other proteins, or less than about 5%other proteins. When the peptide is recombinantly produced, it can alsobe substantially free of culture medium, i.e., culture medium representsless than about 20% of the volume of the protein preparation.

The language “substantially free of chemical precursors or otherchemicals” includes preparations of the peptide in which it is separatedfrom chemical precursors or other chemicals that are involved in itssynthesis. In one embodiment, the language “substantially free ofchemical precursors or other chemicals” includes preparations of thenuclear hormone receptor peptide having less than about 30% (by dryweight) chemical precursors or other chemicals, less than about 20%chemical precursors or other chemicals, less than about 10% chemicalprecursors or other chemicals, or less than about 5% chemical precursorsor other chemicals.

The isolated nuclear hormone receptor peptide can be purified from cellsthat naturally express it, purified from cells that have been altered toexpress it (recombinant), or synthesized using known protein synthesismethods. Experimental data as provided in FIG. 1 indicates that nuclearhormone receptors of the present invention are expressed in humans inbreast, brain, head-neck tissue, testis, placenta, retina, liver,kidney, HeLa cell tissue, and fetal liver-spleen. For example, a nucleicacid molecule encoding the nuclear hormone receptor peptide is clonedinto an expression vector, the expression vector introduced into a hostcell and the protein expressed in the host cell. The protein can then beisolated from the cells by an appropriate purification scheme usingstandard protein purification techniques. Many of these techniques aredescribed in detail below.

Accordingly, the present invention provides proteins that consist of theamino acid sequences provided in FIG. 2 (SEQ ID NO:2), for example,proteins encoded by the transcript/cDNA nucleic acid sequences shown inFIG. 1 (SEQ ID NO:1) and the genomic sequences provided in FIG. 3 (SEQID NO:3). The amino acid sequence of such a protein is provided in FIG.2. A protein consists of an amino acid sequence when the amino acidsequence is the final amino acid sequence of the protein.

The present invention further provides proteins that consist essentiallyof the amino acid sequences provided in FIG. 2 (SEQ ID NO:2), forexample, proteins encoded by the transcript/cDNA nucleic acid sequencesshown in FIG. 1 (SEQ ID NO:1) and the genomic sequences provided in FIG.3 (SEQ ID NO:3). A protein consists essentially of an amino acidsequence when such an amino acid sequence is present with only a fewadditional amino acid residues, for example from about 1 to about 100 orso additional residues, typically from 1 to about 20 additional residuesin the final protein.

The present invention further provides proteins that comprise the aminoacid sequences provided in FIG. 2 (SEQ ID NO:2), for example, proteinsencoded by the transcript/cDNA nucleic acid sequences shown in FIG. 1(SEQ ID NO:1) and the genomic sequences provided in FIG. 3 (SEQ IDNO:3). A protein comprises an amino acid sequence when the amino acidsequence is at least part of the final amino acid sequence of theprotein. In such a fashion, the protein can be only the peptide or haveadditional amino acid molecules, such as amino acid residues (contiguousencoded sequence) that are naturally associated with it or heterologousamino acid residues/peptide sequences. Such a protein can have a fewadditional amino acid residues or can comprise several hundred or moreadditional amino acids. The preferred classes of proteins that arecomprised of the nuclear hormone receptor peptides of the presentinvention are the naturally occurring mature proteins. A briefdescription of how various types of these proteins can be made/isolatedis provided below.

The nuclear hormone receptor peptides of the present invention can beattached to heterologous sequences to form chimeric or fusion proteins.Such chimeric and fusion proteins comprise a nuclear hormone receptorpeptide operatively linked to a heterologous protein having an aminoacid sequence not substantially homologous to the nuclear hormonereceptor peptide. “Operatively linked” indicates that the nuclearhormone receptor peptide and the heterologous protein are fusedin-frame. The heterologous protein can be fused to the N-terminus orC-terminus of the nuclear hormone receptor peptide.

In some uses, the fusion protein does not affect the activity of thenuclear hormone receptor peptide per se. For example, the fusion proteincan include, but is not limited to, enzymatic fusion proteins, forexample beta-galactosidase fusions, yeast two-hybrid GAL fusions,poly-His fusions, MYC-tagged, HI-tagged and Ig fusions. Such fusionproteins, particularly poly-His fusions, can facilitate the purificationof recombinant nuclear hormone receptor peptide. In certain host cells(e.g., mammalian host cells), expression and/or secretion of a proteincan be increased by using a heterologous signal sequence.

A chimeric or fusion protein can be produced by standard recombinant DNAtechniques. For example, DNA fragments coding for the different proteinsequences are ligated together in-frame in accordance with conventionaltechniques. In another embodiment, the fusion gene can be synthesized byconventional techniques including automated DNA synthesizers.Alternatively, PCR amplification of gene fragments can be carried outusing anchor primers which give rise to complementary overhangs betweentwo consecutive gene fragments which can subsequently be annealed andre-amplified to generate a chimeric gene sequence (see Ausubel et al.,Current Protocols in Molecular Biology, 1992). Moreover, many expressionvectors are commercially available that already encode a fusion moiety(e.g., a GST protein). A nuclear hormone receptor peptide-encodingnucleic acid can be cloned into such an expression vector such that thefusion moiety is linked in-frame to the nuclear hormone receptorpeptide.

As mentioned above, the present invention also provides and enablesobvious variants of the amino acid sequence of the proteins of thepresent invention, such as naturally occurring mature forms of thepeptide, allelic/sequence variants of the peptides, non-naturallyoccurring recombinantly derived variants of the peptides, and orthologsand paralogs of the peptides. Such variants can readily be generatedusing art-known techniques in the fields of recombinant nucleic acidtechnology and protein biochemistry. It is understood, however, thatvariants exclude any amino acid sequences disclosed prior to theinvention.

Such variants can readily be identified/made using molecular techniquesand the sequence information disclosed herein. Further, such variantscan readily be distinguished from other peptides based on sequenceand/or structural homology to the nuclear hormone receptor peptides ofthe present invention. The degree of homology/identity present will bebased primarily on whether the peptide is a functional variant ornon-functional variant, the amount of divergence present in the paralogfamily and the evolutionary distance between the orthologs.

To determine the percent identity of two amino acid sequences or twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes). Ina preferred embodiment, at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% ormore of the length of a reference sequence is aligned for comparisonpurposes. The amino acid residues or nucleotides at corresponding aminoacid positions or nucleotide positions are then compared. When aposition in the first sequence is occupied by the same amino acidresidue or nucleotide as the corresponding position in the secondsequence, then the molecules are identical at that position (as usedherein amino acid or nucleic acid “identity” is equivalent to amino acidor nucleic acid “homology”). The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which need to be introduced for optimal alignment of the twosequences.

The comparison of sequences and determination of percent identity andsimilarity between two sequences can be accomplished using amathematical algorithm. (Computational Molecular Biology, Lesk, A. M.,ed., Oxford University Press, New York, 1988; Biocomputing: Informaticsand Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991). In a preferred embodiment, the percent identity betweentwo amino acid sequences is determined using the Needleman and Wunsch(J. Mol. Biol. (48):444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package (availableat http://www.gcg.com), using either a Blossom 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment thepercent identity between two nucleotide sequences is determined usingthe GAP program in the GCG software package (Devereux, J., et al.,Nucleic Acids Res. 12(1):387 (1984)) (available at http://www.gcg.com),using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, thepercent identity between two amino acid or nucleotide sequences isdetermined using the algorithm of E. Myers and W. Miller (CABIOS,4:11-17 (1989)) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4.

The nucleic acid and protein sequences of the present invention canfurther be used as a “query sequence” to perform a search againstsequence databases to, for example, identify other family members orrelated sequences. Such searches can be performed using the NBLAST andXBLAST programs (version 2.0) of Altschul, et al. (J. Mol. Biol.215:403-10 (1990)). BLAST nucleotide searches can be performed with theNBLAST program, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to the nucleic acid molecules of the invention. BLAST proteinsearches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to the proteinsof the invention. To obtain gapped alignments for comparison purposes,Gapped BLAST can be utilized as described in Altschul et al. (NucleicAcids Res. 25(17):3389-3402 (1997)). When utilizing BLAST and gappedBLAST programs, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used.

Full-length pre-processed forms, as well as mature processed forms, ofproteins that comprise one of the peptides of the present invention canreadily be identified as having complete sequence identity to one of thenuclear hormone receptor peptides of the present invention as well asbeing encoded by the same genetic locus as the nuclear hormone receptorpeptide provided herein. As indicated by the data presented in FIG. 3,the nuclear hormone receptor of the present invention was determined tobe located on a Bacteria Artificial Chromosome (“BAC”), Bac AccessionAC018629, which is known to be on chromosome 17.

Allelic variants of a nuclear hormone receptor peptide can readily beidentified as being a human protein having a high degree (significant)of sequence homology/identity to at least a portion of the nuclearhormone receptor peptide as well as being encoded by the same geneticlocus as the nuclear hormone receptor peptide provided herein. Geneticlocus can readily be determined based on the genomic informationprovided in FIG. 3, such as the genomic sequence mapped to the referencehuman. As indicated by the data presented in FIG. 3, the nuclear hormonereceptor of the present invention was determined to be located on aBacteria Artificial Chromosome (‘BAC’), Bac Accession AC018629, which isknown to be on chromosome 17. As used herein, two proteins (or a regionof the proteins) have significant homology when the amino acid sequencesare typically at least about 70-80%, 80-90%, and more typically at leastabout 90-95% or more homologous. A significantly homologous amino acidsequence, according to the present invention, will be encoded by anucleic acid sequence that will hybridize to a nuclear hormone receptorpeptide encoding nucleic acid molecule under stringent conditions asmore fully described below.

FIG. 3 provides SNP information that has been found in a gene encodingthe nuclear hormone proteins of the present invention. The followingvariations were seen: C4084G, G6482A, C8066G, T8699C, C12897T, andC14442T. The changes in the amino acid that these SNPs cause can readilybe determined using the universal genetic code and the protein sequenceprovided in FIG. 2 as a base.

Paralogs of a nuclear hormone receptor peptide can readily be identifiedas having some degree of significant sequence homology/identity to atleast a portion of the nuclear hormone receptor peptide, as beingencoded by a gene from humans, and as having similar activity orfunction. Two proteins will typically be considered paralogs when theamino acid sequences are typically at least about 60% or greater, andmore typically at least about 70% or greater homology through a givenregion or domain. Such paralogs will be encoded by a nucleic acidsequence that will hybridize to a nuclear hormone receptor peptideencoding nucleic acid molecule under moderate to stringent conditions asmore fully described below.

Orthologs of a nuclear hormone receptor peptide can readily beidentified as having some degree of significant sequencehomology/identity to at least a portion of the nuclear hormone receptorpeptide as well as being encoded by a gene from another organism.Preferred orthologs will be isolated from mammals, preferably primates,for the development of human therapeutic targets and agents. Suchorthologs will be encoded by a nucleic acid sequence that will hybridizeto a nuclear hormone receptor peptide encoding nucleic acid moleculeunder moderate to stringent conditions, as more fully described below,depending on the degree of relatedness of the two organisms yielding theproteins.

Non-naturally occurring variants of the nuclear hormone receptorpeptides of the present invention can readily be generated usingrecombinant techniques. Such variants include, but are not limited todeletions, additions and substitutions in the amino acid sequence of thenuclear hormone receptor peptide. For example, one class ofsubstitutions are conserved amino acid substitution. Such substitutionsare those that substitute a given amino acid in a nuclear hormonereceptor peptide by another amino acid of like characteristics.Typically seen as conservative substitutions are the replacements, onefor another, among the aliphatic amino acids Ala, Val, Leu, and Ile;interchange of the hydroxyl residues Ser and Thr; exchange of the acidicresidues Asp and Glu; substitution between the amide residues Asn andGln; exchange of the basic residues Lys and Arg; and replacements amongthe aromatic residues Phe and Tyr. Guidance concerning which amino acidchanges are likely to be phenotypically silent are found in Bowie etal., Science 247:1306-1310 (1990).

Variant nuclear hormone receptor peptides can be fully functional or canlack function in one or more activities, e.g. ability to bind substrate,ability to phosphorylate substrate, ability to mediate signaling, etc.Fully functional variants typically contain only conservative variationor variation in non-critical residues or in non-critical regions. FIG. 2provides the result of protein analysis and can be used to identifycritical domains/regions. Functional variants can also containsubstitution of similar amino acids that result in no change or aninsignificant change in function. Alternatively, such substitutions maypositively or negatively affect function to some degree.

Non-functional variants typically contain one or more non-conservativeamino acid substitutions, deletions, insertions, inversions, ortruncation or a substitution, insertion, inversion, or deletion in acritical residue or critical region.

Amino acids that are essential for function can be identified by methodsknown in the art, such as site-directed mutagenesis or alanine-scanningmutagenesis (Cunningham et al., Science 244:1081-1085 (1989)),particularly using the results provided in FIG. 2. The latter procedureintroduces single alanine mutations at every residue in the molecule.The resulting mutant molecules are then tested for biological activitysuch as nuclear hormone receptor activity or in assays such as an invitro proliferative activity. Sites that are critical for bindingpartner/substrate binding can also be determined by structural analysissuch as crystallization, nuclear magnetic resonance or photoaffinitylabeling (Smith et al., J. Mol. Biol. 224:899-904 (1992); de Vos et al,Science 255:306-312 (1992)).

The present invention further provides fragments of the nuclear hormonereceptor peptides, in addition to proteins and peptides that compriseand consist of such fragments, particularly those comprising theresidues identified in FIG. 2. The fragments to which the inventionpertains, however, are not to be construed as encompassing fragmentsthat may be disclosed publicly prior to the present invention.

As used herein, a fragment comprises at least 8, 10, 12, 14, 16, or morecontiguous amino acid residues from a nuclear hormone receptor peptide.Such fragments can be chosen based on the ability to retain one or moreof the biological activities of the nuclear hormone receptor peptide orcould be chosen for the ability to perform a function, e.g. bind asubstrate or act as an immunogen. Particularly important fragments arebiologically active fragments, peptides that are, for example, about 8or more amino acids in length. Such fragments will typically comprise adomain or motif of the nuclear hormone receptor peptide, e.g., activesite, a transmembrane domain or a substrate-binding domain. Further,possible fragments include, but are not limited to, domain or motifcontaining fragments, soluble peptide fragments, and fragmentscontaining immunogenic structures. Predicted domains and functionalsites are readily identifiable by computer programs well known andreadily available to those of skill in the art (e.g., PROSITE analysis).The results of one such analysis are provided in FIG. 2.

Polypeptides often contain amino acids other than the 20 amino acidscommonly referred to as the 20 naturally occurring amino acids. Further,many amino acids, including the terminal amino acids, may be modified bynatural processes, such as processing and other post-translationalmodifications, or by chemical modification techniques well known in theart. Common modifications that occur naturally in nuclear hormonereceptor peptides are described in basic texts, detailed monographs, andthe research literature, and they are well known to those of skill inthe art (some of these features are identified in FIG. 2).

Known modifications include, but are not limited to, acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent crosslinks, formation of cystine, formation ofpyroglutamate, formylation, gamma carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination.

Such modifications are well known to those of skill in the art and havebeen described in great detail in the scientific literature. Severalparticularly common modifications, glycosylation, lipid attachment,sulfation, gamma-carboxylation of glutamic acid residues, hydroxylationand ADP-ribosylation, for instance, are described in most basic texts,such as Proteins-Structure and Molecular Properties, 2nd Ed., T. E.Creighton, W. H. Freeman and Company, New York (1993). Many detailedreviews are available on this subject, such as by Wold, F.,Posttranslational Covalent Modification of proteins, B. C. Johnson, Ed.,Academic Press, New York 1-12 (1983); Seifter et al. (Meth. Enzymol.182: 626-646 (1990)) and Rattan et al. (Ann N.Y. Acad. Sci. 663:48-62(1992)).

Accordingly, the nuclear hormone receptor peptides of the presentinvention also encompass derivatives or analogs in which a substitutedamino acid residue is not one encoded by the genetic code, in which asubstituent group is included, in which the mature nuclear hormonereceptor peptide is fused with another compound, such as a compound toincrease the half-life of the nuclear hormone receptor peptide (forexample, polyethylene glycol), or in which the additional amino acidsare fused to the mature nuclear hormone receptor peptide, such as aleader or secretory sequence or a sequence for purification of themature nuclear hormone receptor peptide or a pro-protein sequence.

Protein/Pentide Uses

The proteins of the present invention can be used in substantial andspecific assays related to the fractional information provided in theFigures; to raise antibodies or to elicit another immune response; as areagent (including the labeled reagent) in assays designed toquantitatively determine levels of the protein (or its binding partneror ligand) in biological fluids; and as markers for tissues in which thecorresponding protein is preferentially expressed (either constitutivelyor at a particular stage of tissue differentiation or development or ina disease state). Where the protein binds or potentially binds toanother protein or ligand (such as, for example, in a nuclear hormonereceptor-effector protein interaction or nuclear hormone receptor-ligandinteraction), the protein can be used to identify the bindingpartner/ligand so as to develop a system to identify inhibitors of thebinding interaction. Any or all of these uses are capable of beingdeveloped into reagent grade or kit format for commercialization ascommercial products.

Methods for performing the uses listed above are well known to thoseskilled in the art. References disclosing such methods include“Molecular Cloning: A Laboratory Manual”, 2d ed., Cold Spring HarborLaboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatis eds.,1989, and “Methods in Enzymology: Guide to Molecular CloningTechniques”, Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987.

The potential uses of the peptides of the present invention are basedprimarily on the source of the protein as well as the class/action ofthe protein. For example, nuclear hormone receptors isolated from humansand their human/mammalian orthologs serve as targets for identifyingagents for use in mammalian therapeutic applications, e.g. a human drug,particularly in modulating a biological or pathological response in acell or tissue that expresses the nuclear hormone receptor. Experimentaldata as provided in FIG. 1 indicates that nuclear hormone receptors ofthe present invention are expressed in humans in breast, brain,head-neck tissue, testis, placenta, retina, liver, kidney, HeLa celltissue, and fetal liver-spleen. Specifically, a virtual northern blotshows expression in breast, testis, placenta, retina, head-neck tissue,and fetal liver-spleen. In addition, PCR-based tissue screening panelsindicate expression in brain, placenta, liver, kidney, and HeLa celltissue. A large percentage of pharmaceutical agents are being developedthat modulate the activity of nuclear hormone receptor proteins,particularly members of the retinoic acid receptor subfamily (seeBackground of the Invention). The structural and functional informationprovided in the Background and Figures provide specific and substantialuses for the molecules of the present invention, particularly incombination with the expression information provided in FIG. 1.Experimental data as provided in FIG. 1 indicates that nuclear hormonereceptors of the present invention are expressed in humans in breast,brain, head-neck tissue, testis, placenta, retina, liver, kidney, HeLacell tissue, and fetal liver-spleen. Such uses can readily be determinedusing the information provided herein, that which is known in the art,and routine experimentation.

The proteins of the present invention (including variants and fragmentsthat may have been disclosed prior to the present invention) are usefulfor biological assays related to nuclear hormone receptors that arerelated to members of the retinoic acid receptor subfamily. Such assaysinvolve any of the known nuclear hormone receptor functions oractivities or properties useful for diagnosis and treatment of nuclearhormone receptor-related conditions that are specific for the subfamilyof nuclear hormone receptors that the one of the present inventionbelongs to, particularly in cells and tissues that express the nuclearhormone receptor. Experimental data as provided in FIG. 1 indicates thatnuclear hormone receptors of the present invention are expressed inhumans in breast, brain, head-neck tissue, testis, placenta, retina,liver, kidney, HeLa cell tissue, and fetal liver-spleen. Specifically, avirtual northern blot shows expression in breast, testis, placenta,retina, head-neck tissue, and fetal liver-spleen. In addition, PCR-basedtissue screening panels indicate expression in brain, placenta, liver,kidney, and HeLa cell tissue.

The proteins of the present invention are also useful in drug screeningassays, in cell-based or cell-free systems. Cell-based systems can benative, i.e., cells that normally express the nuclear hormone receptor,as a biopsy or expanded in cell culture. Experimental data as providedin FIG. 1 indicates that nuclear hormone receptors of the presentinvention are expressed in humans in breast, brain, head-neck tissue,testis, placenta, retina, liver, kidney, HeLa cell tissue, and fetalliver-spleen. In an alternate embodiment, cell-based assays involverecombinant host cells expressing the nuclear hormone receptor protein.

The polypeptides can be used to identify compounds that modulate nuclearhormone receptor activity of the protein in its natural state or analtered form that causes a specific disease or pathology associated withthe nuclear hormone receptor. Both the nuclear hormone receptors of thepresent invention and appropriate variants and fragments can be used inhigh-throughput screens to assay candidate compounds for the ability tobind to the nuclear hormone receptor. These compounds can be furtherscreened against a functional nuclear hormone receptor to determine theeffect of the compound on the nuclear hormone receptor activity.Further, these compounds can be tested in animal or invertebrate systemsto determine activity/effectiveness. Compounds can be identified thatactivate (agonist) or inactivate (antagonist) the nuclear hormonereceptor to a desired degree.

Further, the proteins of the present invention can be used to screen acompound for the ability to stimulate or inhibit interaction between thenuclear hormone receptor protein and a molecule that normally interactswith the nuclear hormone receptor protein, e.g. a substrate or acomponent of the signal pathway that the nuclear hormone receptorprotein normally interacts (for example, another nuclear hormonereceptor). Such assays typically include the steps of combining thenuclear hormone receptor protein with a candidate compound underconditions that allow the nuclear hormone receptor protein, or fragment,to interact with the target molecule, and to detect the formation of acomplex between the protein and the target or to detect the biochemicalconsequence of the interaction with the nuclear hormone receptor proteinand the target, such as any of the associated effects of signaltransduction such as protein phosphorylation, cAMP turnover, andadenylate cyclase activation, etc.

Candidate compounds include, for example, 1) peptides such as solublepeptides, including Ig-tailed fusion peptides and members of randompeptide libraries (see, e.g., Lam et al., Nature 354:82-84 (1991);Houghten et al., Nature 354:84-86 (1991)) and combinatorialchemistry-derived molecular libraries made of D- and/or L-configurationamino acids; 2) phosphopeptides (e.g., members of random and partiallydegenerate, directed phosphopeptide libraries, see, e.g., Song yang etal., Cell 72:767-778 (1993)); 3) antibodies (e.g., polyclonal,monoclonal, humanized, anti-idiotypic, chimeric, and single chainantibodies as well as Fab, F(ab′)₂, Fab expression library fragments,and epitope-binding fragments of antibodies); and 4) small organic andinorganic molecules (e.g., molecules obtained from combinatorial andnatural product libraries).

One candidate compound is a soluble fragment of the receptor thatcompetes for substrate binding. Other candidate compounds include mutantnuclear hormone receptors or appropriate fragments containing mutationsthat affect nuclear hormone receptor function and thus compete forsubstrate. Accordingly, a fragment that competes for substrate, forexample with a higher affinity, or a fragment that binds substrate butdoes not allow release, is encompassed by the invention.

The invention further includes other end point assays to identifycompounds that modulate (stimulate or inhibit) nuclear hormone receptoractivity. The assays typically involve an assay of events in the signaltransduction pathway that indicate nuclear hormone receptor activity.Thus, the phosphorylation of a substrate, activation of a protein, achange in the expression of genes that are up or down-regulated inresponse to the nuclear hormone receptor protein dependent signalcascade can be assayed.

Any of the biological or biochemical functions mediated by the nuclearhormone receptor can be used as an endpoint assay. These include all ofthe biochemical or biochemical/biological events described herein, inthe references cited herein, incorporated by reference for theseendpoint assay targets, and other functions known to those of ordinaryskill in the art or that can be readily identified using the informationprovided in the Figures, particularly FIG. 2. Specifically, a biologicalfunction of a cell or tissues that expresses the nuclear hormonereceptor can be assayed. Experimental data as provided in FIG. 1indicates that nuclear hormone receptors of the present invention areexpressed in humans in breast, brain, head-neck tissue, testis,placenta, retina, liver, kidney, HeLa cell tissue, and fetalliver-spleen. Specifically, a virtual northern blot shows expression inbreast, testis, placenta, retina, head-neck tissue, and fetalliver-spleen. In addition, PCR-based tissue screening panels indicateexpression in brain, placenta, liver, kidney, and HeLa cell tissue.

Binding and/or activating compounds can also be screened by usingchimeric nuclear hormone receptor proteins in which the amino terminalextracellular domain, or parts thereof, the entire transmembrane domainor subregions, such as any of the seven transmembrane segments or any ofthe intracellular or extracellular loops and the carboxy terminalintracellular domain, or parts thereof, can be replaced by heterologousdomains or subregions. For example, a substrate-binding region can beused that interacts with a different substrate then that which isrecognized by the native nuclear hormone receptor. Accordingly, adifferent set of signal transduction components is available as anend-point assay for activation. This allows for assays to be performedin other than the specific host cell from which the nuclear hormonereceptor is derived.

The proteins of the present invention are also useful in competitionbinding assays in methods designed to discover compounds that interactwith the nuclear hormone receptor (e.g. binding partners and/orligands). Thus, a compound is exposed to a nuclear hormone receptorpolypeptide under conditions that allow the compound to bind or tootherwise interact with the polypeptide. Soluble nuclear hormonereceptor polypeptide is also added to the mixture. If the test compoundinteracts with the soluble nuclear hormone receptor polypeptide, itdecreases the amount of complex formed or activity from the nuclearhormone receptor target. This type of assay is particularly useful incases in which compounds are sought that interact with specific regionsof the nuclear hormone receptor. Thus, the soluble polypeptide thatcompetes with the target nuclear hormone receptor region is designed tocontain peptide sequences corresponding to the region of interest.

To perform cell free drug screening assays, it is sometimes desirable toimmobilize either the nuclear hormone receptor protein, or fragment, orits target molecule to facilitate separation of complexes fromuncomplexed forms of one or both of the proteins, as well as toaccommodate automation of the assay.

Techniques for immobilizing proteins on matrices can be used in the drugscreening assays. In one embodiment, a fusion protein can be providedwhich adds a domain that allows the protein to be bound to a matrix. Forexample, glutathione-S-transferase fusion proteins can be adsorbed ontoglutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtitre plates, which are then combined withthe cell lysates (e.g., ³⁵S-labeled) and the candidate compound, and themixture incubated under conditions conducive to complex formation (e.g.,at physiological conditions for salt and pH). Following incubation, thebeads are washed to remove any unbound label, and the matrix immobilizedand radiolabel determined directly, or in the supernatant after thecomplexes are dissociated. Alternatively, the complexes can bedissociated from the matrix, separated by SDS-PAGE, and the level ofnuclear hormone receptor-binding protein found in the bead fractionquantitated from the gel using standard electrophoretic techniques. Forexample, either the polypeptide or its target molecule can beimmobilized utilizing conjugation of biotin and streptavidin usingtechniques well known in the art. Alternatively, antibodies reactivewith the protein but which do not interfere with binding of the proteinto its target molecule can be derivatized to the wells of the plate, andthe protein trapped in the wells by antibody conjugation. Preparationsof a nuclear hormone receptor-binding protein and a candidate compoundare incubated in the nuclear hormone receptor protein-presenting wellsand the amount of complex trapped in the well can be quantitated.Methods for detecting such complexes, in addition to those describedabove for the GST-immobilized complexes, include immunodetection ofcomplexes using antibodies reactive with the nuclear hormone receptorprotein target molecule, or which are reactive with nuclear hormonereceptor protein and compete with the target molecule, as well asenzyme-linked assays which rely on detecting an enzymatic activityassociated with the target molecule.

Agents that modulate one of the nuclear hormone receptors of the presentinvention can be identified using one or more of the above assays, aloneor in combination. It is generally preferable to use a cell-based orcell free system first and then confirm activity in an animal or othermodel system. Such model systems are well known in the art and canreadily be employed in this context.

Modulators of nuclear hormone receptor protein activity identifiedaccording to these drug screening assays can be used to treat a subjectwith a disorder mediated by the nuclear hormone receptor pathway, bytreating cells or tissues that express the nuclear hormone receptor.Experimental data as provided in FIG. 1 indicates that nuclear hormonereceptors of the present invention are expressed in humans in breast,brain, head-neck tissue, testis, placenta, retina, liver, kidney, HeLacell tissue, and fetal liver-spleen. These methods of treatment includethe steps of administering a modulator of nuclear hormone receptoractivity in a pharmaceutical composition to a subject in need of suchtreatment, the modulator being identified as described herein.

In yet another aspect of the invention, the nuclear hormone receptorproteins can be used as “bait proteins” in a two-hybrid assay orthree-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al.(1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem.268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; Iwabuchiet al. (1993) Oncogene 8:1693-1696; and Brent WO94/10300), to identifyother proteins, which bind to or interact with the nuclear hormonereceptor and are involved in nuclear hormone receptor activity. Suchnuclear hormone receptor-binding proteins are also likely to be involvedin the propagation of signals by the nuclear hormone receptor proteinsor nuclear hormone receptor targets as, for example, downstream elementsof a nuclear hormone receptor-mediated signaling pathway. Alternatively,such nuclear hormone receptor-binding proteins are likely to be nuclearhormone receptor inhibitors.

The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for a nuclear hormonereceptor protein is fused to a gene encoding the DNA binding domain of aknown transcription factor (e.g., GAL-4). In the other construct, a DNAsequence, from a library of DNA sequences, that encodes an unidentifiedprotein (“prey” or “sample”) is fused to a gene that codes for theactivation domain of the known transcription factor. If the “bait” andthe “prey” proteins are able to interact, in vivo, forming a nuclearhormone receptor-dependent complex, the DNA-binding and activationdomains of the transcription factor are brought into close proximity.This proximity allows transcription of a reporter gene (e.g., LacZ)which is operably linked to a transcriptional regulatory site responsiveto the transcription factor. Expression of the reporter gene can bedetected and cell colonies containing the functional transcriptionfactor can be isolated and used to obtain the cloned gene which encodesthe protein which interacts with the nuclear hormone receptor protein.

This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein inan appropriate animal model. For example, an agent identified asdescribed herein (e.g., a nuclear hormone receptor-modulating agent, anantisense nuclear hormone receptor nucleic acid molecule, a nuclearhormone receptor-specific antibody, or a nuclear hormonereceptor-binding partner) can be used in an animal or other model todetermine the efficacy, toxicity, or side effects of treatment with suchan agent. Alternatively, an agent identified as described herein can beused in an animal or other model to determine the mechanism of action ofsuch an agent. Furthermore, this invention pertains to uses of novelagents identified by the above-described screening assays for treatmentsas described herein.

The nuclear hormone receptor proteins of the present invention are alsouseful to provide a target for diagnosing a disease or predisposition todisease mediated by the peptide. Accordingly, the invention providesmethods for detecting the presence, or levels of, the protein (orencoding mRNA) in a cell, tissue, or organism. Experimental data asprovided in FIG. 1 indicates that nuclear hormone receptors of thepresent invention are expressed in humans in breast, brain, head-necktissue, testis, placenta, retina, liver, kidney, HeLa cell tissue, andfetal liver-spleen. The method involves contacting a biological samplewith a compound capable of interacting with the nuclear hormone receptorprotein such that the interaction can be detected. Such an assay can beprovided in a single detection formal or a multi-detection format suchas an antibody chip array.

One agent for detecting a protein in a sample is an antibody capable ofselectively binding to protein. A biological sample includes tissues,cells and biological fluids isolated from a subject, as well as tissues,cells and fluids present within a subject.

The peptides of the present invention also provide targets fordiagnosing active protein activity, disease, or predisposition todisease, in a patient having a variant peptide, particularly activitiesand conditions that are known for other members of the family ofproteins to which the present one belongs. Thus, the peptide can beisolated from a biological sample and assayed for the presence of agenetic mutation that results in aberrant peptide. This includes aminoacid substitution, deletion, insertion, rearrangement, (as the result ofaberrant splicing events), and inappropriate post-translationalmodification. Analytic methods include altered electrophoretic mobility,altered tryptic peptide digest, altered nuclear hormone receptoractivity in cell-based or cell-free assay, alteration in a substrate orantibody-binding pattern, altered isoelectric point, direct amino acidsequencing, and any other of the known assay techniques useful fordetecting mutations in a protein. Such an assay can be provided in asingle detection format or a multi-detection format such as an antibodychip array.

In vitro techniques for detection of peptide include enzyme linkedimmunosorbent assays (ELISAs), Western blots, immunoprecipitations andimmunofluorescence using a detection reagent, such as an antibody orprotein binding agent. Alternatively, the peptide can be detected invivo in a subject by introducing into the subject a labeled anti-peptideantibody or other types of detection agent. For example, the antibodycan be labeled with a radioactive marker whose presence and location ina subject can be detected by standard imaging techniques. Particularlyuseful are methods that detect the allelic variant of a peptideexpressed in a subject and methods which detect fragments of a peptidein a sample.

The peptides are also useful in pharmacogenomic analysis.Pharmacogenomics deal with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See, e.g., Eichelbaum, M. (Clin. Exp.Pharmacol. Physiol. 23(10-11):983-985 (1996)), and Linder, M. W. (Clin.Chem. 43(2):254-266 (1997)). The clinical outcomes of these variationsresult in severe toxicity of therapeutic drugs in certain individuals ortherapeutic failure of drugs in certain individuals as a result ofindividual variation in metabolism. Thus, the genotype of the individualcan determine the way a therapeutic compound acts on the body or the waythe body metabolizes the compound. Further, the activity of drugmetabolizing enzymes effects both the intensity and duration of drugaction. Thus, the pharmacogenomics of the individual permit theselection of effective compounds and effective dosages of such compoundsfor prophylactic or therapeutic treatment based on the individual'sgenotype. The discovery of genetic polymorphisms in some drugmetabolizing enzymes has explained why some patients do not obtain theexpected drug effects, show an exaggerated drug effect, or experienceserious toxicity from standard drug dosages. Polymorphisms can beexpressed in the phenotype of the extensive metabolizer and thephenotype of the poor metabolizer. Accordingly, genetic polymorphism maylead to allelic protein variants of the nuclear hormone receptor proteinin which one or more of the nuclear hormone receptor functions in onepopulation is different from those in another population. The peptidesthus allow a target to ascertain a genetic predisposition that canaffect treatment modality. Thus, in a ligand-based treatment,polymorphism may give rise to amino terminal extracellular domainsand/or other substrate-binding regions that are more or less active insubstrate binding, and nuclear hormone receptor activation. Accordingly,substrate dosage would necessarily be modified to maximize thetherapeutic effect within a given population containing a polymorphism.As an alternative to genotyping, specific polymorphic peptides could beidentified.

The peptides are also useful for treating a disorder characterized by anabsence of, inappropriate, or unwanted expression of the protein.Experimental data as provided in FIG. 1 indicates that nuclear hormonereceptors of the present invention are expressed in humans in breast,brain, head-neck tissue, testis, placenta, retina, liver, kidney, HeLacell tissue, and fetal liver-spleen. Accordingly, methods for treatmentinclude the use of the nuclear hormone receptor protein or fragments.

Antibodies

The invention also provides antibodies that selectively bind to one ofthe peptides of the present invention, a protein comprising such apeptide, as well as variants and fragments thereof. As used herein, anantibody selectively binds a target peptide when it binds the targetpeptide and does not significantly bind to unrelated proteins. Anantibody is still considered to selectively bind a peptide even if italso binds to other proteins that are not substantially homologous withthe target peptide so long as such proteins share homology with afragment or domain of the peptide target of the antibody. In this case,it would be understood that antibody binding to the peptide is stillselective despite some degree of cross-reactivity.

As used herein, an antibody is defined in terms consistent with thatrecognized within the art: they are multi-subunit proteins produced by amammalian organism in response to an antigen challenge. The antibodiesof the present invention include polyclonal antibodies and monoclonalantibodies, as well as fragments of such antibodies, including, but notlimited to, Fab or F(ab′)₂, and Fv fragments.

Many methods are known for generating and/or identifying antibodies to agiven target peptide. Several such methods are described by Harlow,Antibodies, Cold Spring Harbor Press, (1989).

In general, to generate antibodies, an isolated peptide is used as animmunogen and is administered to a mammalian organism, such as a rat,rabbit or mouse. The full-length protein, an antigenic peptide fragmentor a fusion protein can be used. Particularly important fragments arethose covering functional domains, such as the domains identified inFIG. 2, and domain of sequence homology or divergence amongst thefamily, such as those that can readily be identified using proteinalignment methods and as presented in the Figures.

Antibodies are preferably prepared from regions or discrete fragments ofthe nuclear hormone receptor proteins. Antibodies can be prepared fromany region of the peptide as described herein. However, preferredregions will include those involved in function/activity and/or nuclearhormone receptor/binding partner interaction. FIG. 2 can be used toidentify particularly important regions while sequence alignment can beused to identify conserved and unique sequence fragments.

An antigenic fragrant will typically comprise at least 8 contiguousamino acid residues. The antigenic peptide can comprise, however, atleast 10, 12, 14, 16 or more amino acid residues. Such fragments can beselected on a physical property, such as fragments correspond to regionsthat are located on the surface of the protein, e.g., hydrophilicregions or can be selected based on sequence uniqueness (see FIG. 2).

Detection on an antibody of the present invention can be facilitated bycoupling (i.e., physically linking) the antibody to a detectablesubstance. Examples of detectable substances include various enzymes,prosthetic groups, fluorescent materials, luminescent materials,bioluminescent materials, and radioactive materials. Examples ofsuitable enzymes include horseradish peroxidase, alkaline phosphatase,ε-galactosidase, or acetylcholinesterase; examples of suitableprosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin,and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or³H.

Antibody Uses

The antibodies can be used to isolate one of the proteins of the presentinvention by standard techniques, such as affinity chromatography orimmunoprecipitation. The antibodies can facilitate the purification ofthe natural protein from cells and recombinantly produced proteinexpressed in host cells. In addition, such antibodies are useful todetect the presence of one of the proteins of the present invention incells or tissues to determine the pattern of expression of the proteinamong various tissues in an organism and over the course of normaldevelopment. Experimental data as provided in FIG. 1 indicates thatnuclear hormone receptors of the present invention are expressed inhumans in breast, brain, head-neck tissue, testis, placenta, retina,liver, kidney, HeLa cell tissue, and fetal liver-spleen. Specifically, avirtual northern blot shows expression in breast, testis, placenta,retina, head-neck tissue, and fetal liver-spleen. In addition, PCR-basedtissue screening panels indicate expression in brain, placenta, liver,kidney, and HeLa cell tissue. Further, such antibodies can be used todetect protein in situ, in vitro, or in a cell lysate or supernatant inorder to evaluate the abundance and pattern of expression. Also, suchantibodies can be used to assess abnormal tissue distribution orabnormal expression during development or progression of a biologicalcondition. Antibody detection of circulating fragments of the fulllength protein can be used to identify turnover.

Further, the antibodies can be used to assess expression in diseasestates such as in active stages of the disease or in an individual witha predisposition toward disease related to the protein's function. Whena disorder is caused by an inappropriate tissue distribution,developmental expression, level of expression of the protein, orexpressed/processed form, the antibody can be prepared against thenormal protein. Experimental data as provided in FIG. 1 indicates thatnuclear hormone receptors of the present invention are expressed inhumans in breast, brain, head-neck tissue, testis, placenta, retina,liver, kidney, HeLa cell tissue, and fetal liver-spleen. If a disorderis characterized by a specific mutation in the protein, antibodiesspecific for this mutant protein can be used to assay for the presenceof the specific mutant protein.

The antibodies can also be used to assess normal and aberrantsubcellular localization of cells in the various tissues in an organism.Experimental data as provided in FIG. 1 indicates that nuclear hormonereceptors of the present invention are expressed in humans in breast,brain, head-neck tissue, testis, placenta, retina, liver, kidney, HeLacell tissue, and fetal liver-spleen. The diagnostic uses can be applied,not only in genetic testing, but also in monitoring a treatmentmodality. Accordingly, where treatment is ultimately aimed at correctingexpression level or the presence of aberrant sequence and aberranttissue distribution or developmental expression, antibodies directedagainst the protein or relevant fragments can be used to monitortherapeutic efficacy.

Additionally, antibodies are useful in pharmacogenomic analysis. Thus,antibodies prepared against polymorphic proteins can be used to identifyindividuals that require modified treatment modalities. The antibodiesare also useful as diagnostic tools as an immunological marker foraberrant protein analyzed by electrophoretic mobility, isoelectricpoint, tryptic peptide digest, and other physical assays known to thosein the art.

The antibodies are also useful for tissue typing. Experimental data asprovided in FIG. 1 indicates that nuclear hormone receptors of thepresent invention are expressed in humans in breast, brain, head-necktissue, testis, placenta, retina, liver, kidney, HeLa cell tissue, andfetal liver-spleen. Thus, where a specific protein has been correlatedwith expression in a specific tissue, antibodies that are specific forthis protein can be used to identify a tissue type.

The antibodies are also useful for inhibiting protein function, forexample, blocking the binding of the nuclear hormone receptor peptide toa binding partner such as a substrate. These uses can also be applied ina therapeutic context in which treatment involves inhibiting theprotein's function. An antibody can be used, for example, to blockbinding, thus modulating (agonizing or antagonizing) the peptidesactivity. Antibodies can be prepared against specific fragmentscontaining sites required for function or against intact protein that isassociated with a cell or cell membrane. See FIG. 2 for structuralinformation relating to the proteins of the present invention.

The invention also encompasses kits for using antibodies to detect thepresence of a protein in a biological sample. The kit can compriseantibodies such as a labeled or labelable antibody and a compound oragent for detecting protein in a biological sample; means fordetermining the amount of protein in the sample; means for comparing theamount of protein in the sample with a standard; and instructions foruse. Such a kit can be supplied to detect a single protein or epitope orcan be configured to detect one of a multitude of epitopes, such as inan antibody detection array. Arrays are described in detail below fornuleic acid arrays and similar methods have been developed for antibodyarrays.

Nucleic Acid Molecules

The present invention further provides isolated nucleic acid moleculesthat encode a nuclear hormone receptor peptide or protein of the presentinvention (cDNA, transcript and genomic sequence). Such nucleic acidmolecules will consist of, consist essentially of, or comprise anucleotide sequence that encodes one of the nuclear hormone receptorpeptides of the present invention, an allelic variant thereof, or anortholog or paralog thereof.

As used herein, an “isolated” nucleic acid molecule is one that isseparated from other nucleic acid present in the natural source of thenucleic acid. Preferably, an “isolated” nucleic acid is free ofsequences which naturally flank the nucleic acid (i.e., sequenceslocated at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA ofthe organism from which the nucleic acid is derived. However, there canbe some flanking nucleotide sequences, for example up to about 5 KB, 4KB, 3 KB, 2 KB, or 1 KB or less, particularly contiguous peptideencoding sequences and peptide encoding sequences within the same genebut separated by introns in the genomic sequence. The important point isthat the nucleic acid is isolated from remote and unimportant flankingsequences such that it can be subjected to the specific manipulationsdescribed herein such as recombinant expression, preparation of probesand primers, and other uses specific to the nucleic acid sequences.

Moreover, an “isolated” nucleic acid molecule, such as a transcript/cDNAmolecule, can be substantially free of other cellular material, orculture medium when produced by recombinant techniques, or chemicalprecursors or other chemicals when chemically synthesized. However, thenucleic acid molecule can be fused to other coding or regulatorysequences and still be considered isolated.

For example, recombinant DNA molecules contained in a vector areconsidered isolated. Further examples of isolated DNA molecules includerecombinant DNA molecules maintained in heterologous host cells orpurified (partially or substantially) DNA molecules in solution.Isolated RNA molecules include in vivo or in vitro RNA transcripts ofthe isolated DNA molecules of the present invention. Isolated nucleicacid molecules according to the present invention further include suchmolecules produced synthetically.

Accordingly, the present invention provides nucleic acid molecules thatconsist of the nucleotide sequence shown in FIG. 1 or 3 (SEQ ID NO:1,transcript sequence and SEQ ID NO:3, genomic sequence), or any nucleicacid molecule that encodes the protein provided in FIG. 2, SEQ ID NO:2.A nucleic acid molecule consists of a nucleotide sequence when thenucleotide sequence is the complete nucleotide sequence of the nucleicacid molecule.

The present invention further provides nucleic acid molecules thatconsist essentially of the nucleotide sequence shown in FIG. 1 or 3 (SEQID NO:1, transcript sequence and SEQ ID NO:3, genomic sequence), or anynucleic acid molecule that encodes the protein provided in FIG. 2, SEQID NO:2. A nucleic acid molecule consists essentially of a nucleotidesequence when such a nucleotide sequence is present with only a fewadditional nucleic acid residues in the final nucleic acid molecule.

The present invention further provides nucleic acid molecules thatcomprise the nucleotide sequences shown in FIG. 1 or 3 (SEQ ID NO:1,transcript sequence and SEQ ID NO:3, genomic sequence), or any nucleicacid molecule that encodes the protein provided in FIG. 2, SEQ ID NO:2.A nucleic acid molecule comprises a nucleotide sequence when thenucleotide sequence is at least part of the final nucleotide sequence ofthe nucleic acid molecule. In such a fashion, the nucleic acid moleculecan be only the nucleotide sequence or have additional nucleic acidresidues, such as nucleic acid residues that are naturally associatedwith it or heterologous nucleotide sequences. Such a nucleic acidmolecule can have a few additional nucleotides or can comprises severalhundred or more additional nucleotides. A brief description of howvarious types of these nucleic acid molecules can be readilymade/isolated is provided below.

In FIGS. 1 and 3, both coding and non-coding sequences are provided.Because of the source of the present invention, humans genomic sequence(FIG. 3) and cDNA/transcript sequences (FIG. 1), the nucleic acidmolecules in the Figures will contain genomic intronic sequences, 5′ and3′ non-coding sequences, gene regulatory regions and non-codingintergenic sequences. In general such sequence features are either notedin FIGS. 1 and 3 or can readily be identified using computational toolsknown in the art. As discussed below, some of the non-coding regions,particularly gene regulatory elements such as promoters, are useful fora variety of purposes, e.g. control of heterologous gene expression,target for identifying gene activity modulating compounds, and areparticularly claimed as fragments of the genomic sequence providedherein.

The isolated nucleic acid molecules can encode the mature protein plusadditional amino or carboxyl-terminal amino acids, or amino acidsinterior to the mature peptide (when the mature form has more than onepeptide chain, for instance). Such sequences may play a role inprocessing of a protein from precursor to a mature form, facilitateprotein trafficking, prolong or shorten protein half-life or facilitatemanipulation of a protein for assay or production, among other things.As generally is the case in situ, the additional amino acids may beprocessed away from the mature protein by cellular enzymes.

As mentioned above, the isolated nucleic acid molecules include, but arenot limited to, the sequence encoding the nuclear hormone receptorpeptide alone, the sequence encoding the mature peptide and additionalcoding sequences, such as a leader or secretory sequence (e.g., apre-pro or pro-protein sequence), the sequence encoding the maturepeptide, with or without the additional coding sequences, plusadditional non-coding sequences, for example introns and non-coding 5′and 3′ sequences such as transcribed but non-translated sequences thatplay a role in transcription, mRNA processing (including splicing andpolyadenylation signals), ribosome binding and stability of mRNA. Inaddition, the nucleic acid molecule may be fused to a marker sequenceencoding, for example, a peptide that facilitates purification.

Isolated nucleic acid molecules can be in the form of RNA, such as mRNA,or in the form DNA, including cDNA and genomic DNA obtained by cloningor produced by chemical synthetic techniques or by a combinationthereof. The nucleic acid, especially DNA, can be double-stranded orsingle-stranded. Single-stranded nucleic acid can be the coding strand(sense strand) or the non-coding strand (anti-sense strand).

The invention further provides nucleic acid molecules that encodefragments of the peptides of the present invention as well as nucleicacid molecules that encode obvious variants of the nuclear hormonereceptor proteins of the present invention that are described above.Such nucleic acid molecules may be naturally occurring, such as allelicvariants (same locus), paralogs (different locus), and orthologs(different organism), or may be constructed by recombinant DNA methodsor by chemical synthesis. Such non-naturally occurring variants may bemade by mutagenesis techniques, including those applied to nucleic acidmolecules, cells, or organisms. Accordingly, as discussed above, thevariants can contain nucleotide substitutions, deletions, inversions andinsertions. Variation can occur in either or both the coding andnon-coding regions. The variations can produce both conservative andnon-conservative amino acid substitutions.

The present invention further provides non-coding fragments of thenucleic acid molecules provided in FIGS. 1 and 3. Preferred non-codingfragments include, but are not limited to, promoter sequences, enhancersequences, gene modulating sequences and gene termination sequences.Such fragments are useful in controlling heterologous gene expressionand in developing screens to identify gene-modulating agents. A promotercan readily be identified as being 5′ to the ATG start site in thegenomic sequence provided in FIG. 3.

A fragment comprises a contiguous nucleotide sequence greater than 12 ormore nucleotides. Further, a fragment could at least 30, 40, 50, 100,250 or 500 nucleotides in length. The length of the fragment will bebased on its intended use. For example, the fragment can encode epitopebearing regions of the peptide, or can be useful as DNA probes andprimers. Such fragments can be isolated using the known nucleotidesequence to synthesize an oligonucleotide probe. A labeled probe canthen be used to screen a cDNA library, genomic DNA library, or mRNA toisolate nucleic acid corresponding to the coding region. Further,primers can be used in PCR reactions to clone specific regions of gene.

A probe/primer typically comprises substantially a purifiedoligonucleotide or oligonucleotide pair. The oligonucleotide typicallycomprises a region of nucleotide sequence that hybridizes understringent conditions to at least about 12, 20, 25, 40, 50 or moreconsecutive nucleotides.

Orthologs, homologs, and allelic variants can be identified usingmethods well known in the art. As described in the Peptide Section,these variants comprise a nucleotide sequence encoding a peptide that istypically 60-70%, 70-80%, 80-90%, and more typically at least about90-95% or more homologous to the nucleotide sequence shown in the Figuresheets or a fragment of this sequence. Such nucleic acid molecules canreadily be identified as being able to hybridize under moderate tostringent conditions, to the nucleotide sequence shown in the Figuresheets or a fragment of the sequence. Allelic variants can readily bedetermined by genetic locus of the encoding gene. As indicated by thedata presented in FIG. 3, the nuclear hormone receptor of the presentinvention was determined to be located on a Bacteria ArtificialChromosome (“BAC”), Bac Accession AC018629, which is known to be onchromosome 17.

FIG. 3 provides SNP information that has been found in a gene encodingthe nuclear hormone proteins of the present invention. The followingvariations were seen: C4084G, G6482A, C8066G, T8699C, C12897T, andC14442T. The changes in the amino acid that these SNPs cause can readilybe determined using the universal genetic code and the protein sequenceprovided in FIG. 2 as a base.

As used herein, the term “hybridizes under stringent conditions” isintended to describe conditions for hybridization and washing underwhich nucleotide sequences encoding a peptide at least 60-70% homologousto each other typically remain hybridized to each other. The conditionscan be such that sequences at least about 60%, at least about 70%, or atleast about 80% or more homologous to each other typically remainhybridized to each other. Such stringent conditions are known to thoseskilled in the art and can be found in Current Protocols in MolecularBiology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. One example ofstringent hybridization conditions are hybridization in 6× sodiumchloride/sodium citrate (SSC) at about 45C, followed by one or morewashes in 0.2×SSC, 0.1% SDS at 50-65C. Examples of moderate to lowstringency hybridization conditions are well known in the art.

Nucleic Acid Molecule Uses

The nucleic acid molecules of the present invention are useful forprobes, primers, chemical intermediates, and in biological assays. Thenucleic acid molecules are useful as a hybridization probe for messengerRNA, transcript/cDNA and genomic DNA to isolate full-length cDNA andgenomic clones encoding the peptide described in FIG. 2 and to isolatecDNA and genomic clones that correspond to variants (alleles, orthologs,etc.) producing the same or related peptides shown in FIG. 2. Asillustrated in FIG. 3, known SNP In variations include C4084G, G6482A,C8066G, T8699C, C12897T, and C14442T.

The probe can correspond to any sequence along the entire length of thenucleic acid molecules provided in the Figures. Accordingly, it could bederived from 5′ noncoding regions, the coding region, and 3′ noncodingregions. However, as discussed, fragments are not to be construed asencompassing fragments disclosed prior to the present invention.

The nucleic acid molecules are also useful as primers for PCR to amplifyany given region of a nucleic acid molecule and are useful to synthesizeantisense molecules of desired length and sequence.

The nucleic acid molecules are also useful for constructing recombinantvectors. Such vectors include expression vectors that express a portionof, or all of, the peptide sequences. Vectors also include insertionvectors, used to integrate into another nucleic acid molecule sequence,such as into the cellular genome, to alter in situ expression of a geneand/or gene product. For example, an endogenous coding sequence can bereplaced via homologous recombination with all or part of the codingregion containing one or more specifically introduced mutations.

The nucleic acid molecules are also useful for expressing antigenicportions of the proteins.

The nucleic acid molecules are also useful as probes for determining thechromosomal positions of the nucleic acid molecules by means of in situhybridization methods. As indicated by the data presented in FIG. 3, thenuclear hormone receptor of the present invention was determined to belocated on a Bacteria Artificial Chromosome (“BAC”), Bac AccessionAC018629, which is known to be on chromosome 17.

The nucleic acid molecules are also useful in making vectors containingthe gene regulatory regions of the nucleic acid molecules of the presentinvention.

The nucleic acid molecules are also useful for designing ribozymescorresponding to all, or a part, of the mRNA produced from the nucleicacid molecules described herein.

The nucleic acid molecules are also useful for making vectors thatexpress part, or all, of the peptides.

The nucleic acid molecules are also useful for constructing host cellsexpressing a part, or all, of the nucleic acid molecules and peptides.

The nucleic acid molecules are also useful for constructing transgenicanimals expressing all, or a part, of the nucleic acid molecules andpeptides.

The nucleic acid molecules are also useful as hybridization probes fordetermining the presence, level, form and distribution of nucleic acidexpression. Experimental data as provided in FIG. 1 indicates thatnuclear hormone receptors of the present invention are expressed inhumans in breast, brain, head-neck tissue, testis, placenta, retina,liver, kidney, HeLa cell tissue, and fetal liver-spleen. Specifically, avirtual northern blot shows expression in breast, testis, placenta,retina, head-neck tissue, and fetal liver-spleen. In addition, PCR-basedtissue screening panels indicate expression in brain, placenta, liver,kidney, and HeLa cell tissue. Accordingly, the probes can be used todetect the presence of, or to determine levels of, a specific nucleicacid molecule in cells, tissues, and in organisms. The nucleic acidwhose level is determined can be DNA or RNA. Accordingly, probescorresponding to the peptides described herein can be used to assessexpression and/or gene copy number in a given cell, tissue, or organism.These uses are relevant for diagnosis of disorders involving an increaseor decrease in nuclear hormone receptor protein expression relative tonormal results.

In vitro techniques for detection of mRNA include Northernhybridizations and in situ hybridizations. In vitro techniques fordetecting DNA includes Southern hybridizations and in situhybridization.

Probes can be used as a part of a diagnostic test kit for identifyingcells or tissues that express a nuclear hormone receptor protein, suchas by measuring a level of a nuclear hormone receptor-encoding nucleicacid in a sample of cells from a subject e.g., mRNA or genomic DNA, ordetermining if a nuclear hormone receptor gene has been mutated.Experimental data as provided in FIG. 1 indicates that nuclear hormonereceptors of the present invention are expressed in humans in breast,brain, head-neck tissue, testis, placenta, retina, liver, kidney, HeLacell tissue, and fetal liver-spleen. Specifically, a virtual northernblot shows expression in breast, testis, placenta, retina, head-necktissue, and fetal liver-spleen. In addition, PCR-based tissue screeningpanels indicate expression in brain, placenta, liver, kidney, and HeLacell tissue.

Nucleic acid expression assays are useful for drug screening to identifycompounds that modulate nuclear hormone receptor nucleic acidexpression.

The invention thus provides a method for identifying a compound that canbe used to treat a disorder associated with nucleic acid expression ofthe nuclear hormone receptor gene, particularly biological andpathological processes that are mediated by the nuclear hormone receptorin cells and tissues that express it. Experimental data as provided inFIG. 1 indicates that nuclear hormone receptors of the present inventionare expressed in humans in breast, brain, head-neck tissue, testis,placenta, retina, liver, kidney, HeLa cell tissue, and fetalliver-spleen. The method typically includes assaying the ability of thecompound to modulate the expression of the nuclear hormone receptornucleic acid and thus identifying a compound that can be used to treat adisorder characterized by undesired nuclear hormone receptor nucleicacid expression. The assays can be performed in cell-based and cell-freesystems. Cell-based assays include cells naturally expressing thenuclear hormone receptor nucleic acid or recombinant cells geneticallyengineered to express specific nucleic acid sequences.

The assay for nuclear hormone receptor nucleic acid expression caninvolve direct assay of nucleic acid levels, such as mRNA levels, or oncollateral compounds involved in the signal pathway. Further, theexpression of genes that are up- or down-regulated in response to thenuclear hormone receptor protein signal pathway can also be assayed. Inthis embodiment the regulatory regions of these genes can be operablylinked to a reporter gene such as luciferase.

Thus, modulators of nuclear hormone receptor gene expression can beidentified in a method wherein a cell is contacted with a candidatecompound and the expression of mRNA determined. The level of expressionof nuclear hormone receptor mRNA in the presence of the candidatecompound is compared to the level of expression of nuclear hormonereceptor mRNA in the absence of the candidate compound. The candidatecompound can then be identified as a modulator of nucleic acidexpression based on this comparison and be used, for example to treat adisorder characterized by aberrant nucleic acid expression. Whenexpression of mRNA is statistically significantly greater in thepresence of the candidate compound than in its absence, the candidatecompound is identified as a stimulator of nucleic acid expression. Whennucleic acid expression is statistically significantly less in thepresence of the candidate compound than in its absence, the candidatecompound is identified as an inhibitor of nucleic acid expression.

The invention further provides methods of treatment, with the nucleicacid as a target, using a compound identified through drug screening asa gene modulator to modulate nuclear hormone receptor nucleic acidexpression in cells and tissues that express the nuclear hormonereceptor. Experimental data as provided in FIG. 1 indicates that nuclearhormone receptors of the present invention are expressed in humans inbreast, brain, head-neck tissue, testis, placenta, retina, liver,kidney, HeLa cell tissue, and fetal liver-spleen. Specifically, avirtual northern blot shows expression in breast, testis, placenta,retina, head-neck tissue, and fetal liver-spleen. In addition, PCR-basedtissue screening panels indicate expression in brain, placenta, liver,kidney, and HeLa cell tissue. Modulation includes both up-regulation(i.e. activation or agonization) or down-regulation (suppression orantagonization) or nucleic acid expression.

Alternatively, a modulator for nuclear hormone receptor nucleic acidexpression can be a small molecule or drug identified using thescreening assays described herein as long as the drug or small moleculeinhibits the nuclear hormone receptor nucleic acid expression in thecells and tissues that express the protein. Experimental data asprovided in FIG. 1 indicates that nuclear hormone receptors of thepresent invention are expressed in humans in breast, brain, head-necktissue, testis, placenta, retina, liver, kidney, HeLa cell tissue, andfetal liver-spleen.

The nucleic acid molecules are also useful for monitoring theeffectiveness of modulating compounds on the expression or activity ofthe nuclear hormone receptor gene in clinical trials or in a treatmentregimen. Thus, the gene expression pattern can serve as a barometer forthe continuing effectiveness of treatment with the compound,particularly with compounds to which a patient can develop resistance.The gene expression pattern can also serve as a marker indicative of aphysiological response of the affected cells to the compound.Accordingly, such monitoring would allow either increased administrationof the compound or the administration of alternative compounds to whichthe patient has not become resistant. Similarly, if the level of nucleicacid expression falls below a desirable level, administration of thecompound could be commensurately decreased.

The nucleic acid molecules are also useful in diagnostic assays forqualitative changes in nuclear hormone receptor nucleic acid expression,and particularly in qualitative changes that lead to pathology. Thenucleic acid molecules can be used to detect mutations in nuclearhormone receptor genes and gene expression products such as mRNA. Thenucleic acid molecules can be used as hybridization probes to detectnaturally occurring genetic mutations in the nuclear hormone receptorgene and thereby to determine whether a subject with the mutation is atrisk for a disorder caused by the mutation. Mutations include deletion,addition, or substitution of one or more nucleotides in the gene,chromosomal rearrangement, such as inversion or transposition,modification of genomic DNA, such as aberrant methylation patterns orchanges in gene copy number, such as amplification. Detection of amutated form of the nuclear hormone receptor gene associated with adysfunction provides a diagnostic tool for an active disease orsusceptibility to disease when the disease results from overexpression,underexpression, or altered expression of a nuclear hormone receptorprotein.

Individuals carrying mutations in the nuclear hormone receptor gene canbe detected at the nucleic acid level by a variety of techniques. FIG. 3provides SNP information that has been found in a gene encoding thenuclear hormone proteins of the present invention. The followingvariations were seen: C4084G, G6482A, C8066G, T8699C, C12897T, andC14442T. The changes in the amino acid that these SNPs cause can readilybe determined using the universal genetic code and the protein sequenceprovided in FIG. 2 as a base. As indicated by the data presented in FIG.3, the nuclear hormone receptor of the present invention was determinedto be located on a Bacteria Artificial Chromosome (“BAC”), Bac AccessionAC018629, which is known to be on chromosome 17. Genomic DNA can beanalyzed directly or can be amplified by using PCR prior to analysis.RNA or cDNA can be used in the same way. In some uses, detection of themutation involves the use of a probe/primer in a polymerase chainreaction (PCR) (see, e.g. U.S. Pat. Nos. 4,683,195 and 4,683,202), suchas anchor PCR or RACE PCR, or, alternatively, in a ligation chainreaction (LCR) (see, e.g., Landegran et al., Science 241:1077-1080(1988); and Nakazawa et al., PNAS 91:360-364 (1994)), the latter ofwhich can be particularly useful for detecting point mutations in thegene (see Abravaya et al., Nucleic Acids Res. 23:675-682 (1995)). Thismethod can include the steps of collecting a sample of cells from apatient, isolating nucleic acid (e.g., genomic, mRNA or both) from thecells of the sample, contacting the nucleic acid sample with one or moreprimers which specifically hybridize to a gene under conditions suchthat hybridization and amplification of the gene (if present) occurs,and detecting the presence or absence of an amplification product, ordetecting the size of the amplification product and comparing the lengthto a control sample. Deletions and insertions can be detected by achange in size of the amplified product compared to the normal genotype.Point mutations can be identified by hybridizing amplified DNA to normalRNA or antisense DNA sequences.

Alternatively, mutations in a nuclear hormone receptor gene can bedirectly identified, for example, by alterations in restriction enzymedigestion patterns determined by gel electrophoresis.

Further, sequence-specific ribozymes (U.S. Pat. No. 5,498,531) can beused to score for the presence of specific mutations by development orloss of a ribozyme cleavage site. Perfectly matched sequences can bedistinguished from mismatched sequences by nuclease cleavage digestionassays or by differences in melting temperature.

Sequence changes at specific locations can also be assessed by nucleaseprotection assays such as RNase and S1 protection or the chemicalcleavage method. Furthermore, sequence differences between a mutantnuclear hormone receptor gene and a wild-type gene can be determined bydirect DNA sequencing. A variety of automated sequencing procedures canbe utilized when performing the diagnostic assays (Naeve, C. W., (1995)Biotechniques 19:448), including sequencing by mass spectrometry (see,e.g., PCT International Publication No. WO 94/16101; Cohen et al., Adv.Chromatogr. 36:127-162 (1996); and Griffin et al., Appl. Biochem.Biotechnol. 38:147-159 (1993)).

Other methods for detecting mutations in the gene include methods inwhich protection from cleavage agents is used to detect mismatched basesin RNA/RNA or RNA/DNA duplexes (Myers et al., Science 230:1242 (1985));Cotton et al., PNAS 85:4397 (1988); Saleeba et al., Meth. Enzymol.217:286-295 (1992)), electrophoretic mobility of mutant and wild typenucleic acid is compared (Orita et al., PNAS 86:2766 (1989); Cotton etal., Mutat. Res. 285:125-144 (1993); and Hayashi et al., Genet. Anal.Tech. Appl. 9:73-79 (1992)), and movement of mutant or wild-typefragments in polyacrylamide gels containing a gradient of denaturant isassayed using denaturing gradient gel electrophoresis (Myers et al.,Nature 313:495 (1985)). Examples of other techniques for detecting pointmutations include selective oligonucleotide hybridization, selectiveamplification, and selective primer extension.

The nucleic acid molecules are also useful for testing an individual fora genotype that while not necessarily causing the disease, neverthelessaffects the treatment modality. Thus, the nucleic acid molecules can beused to study the relationship between an individual's genotype and theindividual's response to a compound used for treatment (pharmacogenomicrelationship). Accordingly, the nucleic acid molecules described hereincan be used to assess the mutation content of the nuclear hormonereceptor gene in an individual in order to select an appropriatecompound or dosage regimen for treatment. FIG. 3 provides SNPinformation that has been found in a gene encoding the nuclear hormoneproteins of the present invention. The following variations were seen:C4084G, G6482A, C8066G, T8699C, C12897T, and C14442T. The changes in theamino acid that these SNPs cause can readily be determined using theuniversal genetic code and the protein sequence provided in FIG. 2 as abase.

Thus nucleic acid molecules displaying genetic variations that affecttreatment provide a diagnostic target that can be used to tailortreatment in an individual. Accordingly, the production of recombinantcells and animals containing these polymorphisms allow effectiveclinical design of treatment compounds and dosage regimens.

The nucleic acid molecules are thus useful as antisense constructs tocontrol nuclear hormone receptor gene expression in cells, tissues, andorganisms. A DNA antisense nucleic acid molecule is designed to becomplementary to a region of the gene involved in transcription,preventing transcription and hence production of nuclear hormonereceptor protein. An antisense RNA or DNA nucleic acid molecule wouldhybridize to the mRNA and thus block translation of mRNA into nuclearhormone receptor protein.

Alternatively, a class of antisense molecules can be used to inactivatemRNA in order to decrease expression of nuclear hormone receptor nucleicacid. Accordingly, these molecules can treat a disorder characterized byabnormal or undesired nuclear hormone receptor nucleic acid expression.This technique involves cleavage by means of ribozymes containingnucleotide sequences complementary to one or more regions in the mRNAthat attenuate the ability of the mRNA to be translated. Possibleregions include coding regions and particularly coding regionscorresponding to the catalytic and other functional activities of thenuclear hormone receptor protein, such as substrate binding.

The nucleic acid molecules also provide vectors for gene therapy inpatients containing cells that are aberrant in nuclear hormone receptorgene expression. Thus, recombinant cells, which include the patient'scells that have been engineered ex vivo and returned to the patient, areintroduced into an individual where the cells produce the desirednuclear hormone receptor protein to treat the individual.

The invention also encompasses kits for detecting the presence of anuclear hormone receptor nucleic acid in a biological sample.Experimental data as provided in FIG. 1 indicates that nuclear hormonereceptors of the present invention are expressed in humans in breast,brain, head-neck tissue, testis, placenta, retina, liver, kidney, HeLacell tissue, and fetal liver-spleen. Specifically, a virtual northernblot shows expression in breast, testis, placenta, retina, head-necktissue, and fetal liver-spleen. In addition, PCR-based tissue screeningpanels indicate expression in brain, placenta, liver, kidney, and HeLacell tissue. For example, the kit can comprise reagents such as alabeled or labelable nucleic acid or agent capable of detecting nuclearhormone receptor nucleic acid in a biological sample; means fordetermining the amount of nuclear hormone receptor nucleic acid in thesample, and means for comparing the amount of nuclear hormone receptornucleic acid in the sample with a standard. The compound or agent can bepackaged in a suitable container. The kit can further compriseinstructions for using the kit to detect nuclear hormone receptorprotein mRNA or DNA.

Nucleic Acid Arrays

The present invention further provides nucleic acid detection kits, suchas arrays or microarrays of nucleic acid molecules that are based on thesequence information provided in FIGS. 1 and 3 (SEQ ID NOS:1 and 3).

As used herein “Arrays” or “Microarrays” refers to an array of distinctpolynucleotides or oligonucleotides synthesized on a substrate, such aspaper, nylon or other type of membrane, filter, chip, glass slide, orany other suitable solid support. In one embodiment, the microarray isprepared and used according to the methods described in U.S. Pat. No.5,837,832, Chee et al., PCT application WO95/11995 (Chee et al.),Lockhart, D. J. et al. (1996; Nat. Biotech. 14: 1675-1680) and Schena,M. et al. (1996; Proc. Natl. Acad. Sci. 93: 10614-10619), all of whichare incorporated herein in their entirety by reference. In otherembodiments, such arrays are produced by the methods described by Brownet al., U.S. Pat. No. 5,807,522.

The microarray or detection kit is preferably composed of a large numberof unique, single-stranded nucleic acid sequences, usually eithersynthetic antisense oligonucleotides or fragments of cDNAs, fixed to asolid support. The oligonucleotides are preferably about 6-60nucleotides in length, more preferably 15-30 nucleotides in length, andmost preferably about 20-25 nucleotides in length. For a certain type ofmicroarray or detection kit, it may be preferable to useoligonucleotides that are only 7-20 nucleotides in length. Themicroarray or detection kit may contain oligonucleotides that cover theknown 5′, or 3′, sequence, sequential oligonucleotides which cover thefull length sequence; or unique oligonucleotides selected fromparticular areas along the length of the sequence. Polynucleotides usedin the microarray or detection kit may be oligonucleotides that arespecific to a gene or genes of interest.

In order to produce oligonucleotides to a known sequence for amicroarray or detection kit, the gene(s) of interest (or an ORFidentified from the contigs of the present invention) is typicallyexamined using a computer algorithm which starts at the 5′ or at the 3′end of the nucleotide sequence. Typical algorithms will then identifyoligomers of defined length that are unique to the gene, have a GCcontent within a range suitable for hybridization, and lack predictedsecondary structure that may interfere with hybridization. In certainsituations it may be appropriate to use pairs of oligonucleotides on amicroarray or detection kit. The “pairs” will be identical, except forone nucleotide that preferably is located in the center of the sequence.The second oligonucleotide in the pair (mismatched by one) serves as acontrol. The number of oligonucleotide pairs may range from two to onemillion. The oligomers are synthesized at designated areas on asubstrate using a light-directed chemical process. The substrate may bepaper, nylon or other type of membrane, filter, chip, glass slide or anyother suitable solid support.

In another aspect, an oligonucleotide may be synthesized on the surfaceof the substrate by using a chemical coupling procedure and an ink jetapplication apparatus, as described in PCT application WO95/251116(Baldeschweiler et al.) which is incorporated herein in its entirety byreference. In another aspect, a “gridded” array analogous to a dot (orslot) blot may be used to arrange and link cDNA fragments oroligonucleotides to the surface of a substrate using a vacuum system,thermal, UV, mechanical or chemical bonding procedures. An array, suchas those described above, may be produced by hand or by using availabledevices (slot blot or dot blot apparatus), materials (any suitable solidsupport), and machines (including robotic instruments), and may contain8, 24, 96, 384, 1536, 6144 or more oligonucleotides, or any other numberbetween two and one million which lends itself to the efficient use ofcommercially available instrumentation.

In order to conduct sample analysis using a microarray or detection kit,the RNA or DNA from a biological sample is made into hybridizationprobes. The mRNA is isolated, and cDNA is produced and used as atemplate to make antisense RNA (aRNA). The aRNA is amplified in thepresence of fluorescent nucleotides, and labeled probes are incubatedwith the microarray or detection kit so that the probe sequenceshybridize to complementary oligonucleotides of the microarray ordetection kit. Incubation conditions are adjusted so that hybridizationoccurs with precise complementary matches or with various degrees ofless complementarity. After removal of nonhybridized probes, a scanneris used to determine the levels and patterns of fluorescence. Thescanned images are examined to determine degree of complementarity andthe relative abundance of each oligonucleotide sequence on themicroarray or detection kit. The biological samples may be obtained fromany bodily fluids (such as blood, urine, saliva, phlegm, gastric juices,etc.), cultured cells, biopsies, or other tissue preparations. Adetection system may be used to measure the absence, presence, andamount of hybridization for all of the distinct sequencessimultaneously. This data may be used for large-scale correlationstudies on the sequences, expression patterns, mutations, variants, orpolymorphisms among samples.

Using such arrays, the present invention provides methods to identifythe expression of the nuclear hormone receptor proteins/peptides of thepresent invention. In detail, such methods comprise incubating a testsample with one or more nucleic acid molecules and assaying for bindingof the nucleic acid molecule with components within the test sample.Such assays will typically involve arrays comprising many genes, atleast one of which is a gene of the present invention and or alleles ofthe nuclear hormone receptor gene of the present invention. FIG. 3provides SNP information that has been found in a gene encoding thenuclear hormone proteins of the present invention. The followingvariations were seen: C4084G, G6482A, C8066G, T8699C, C12897T, andC14442T. The changes in the amino acid that these SNPs cause can readilybe determined using the universal genetic code and the protein sequenceprovided in FIG. 2 as a base.

Conditions for incubating a nucleic acid molecule with a test samplevary. Incubation conditions depend on the format employed in the assay,the detection methods employed, and the type and nature of the nucleicacid molecule used in the assay. One skilled in the art will recognizethat any one of the commonly available hybridization, amplification orarray assay formats can readily be adapted to employ the novel fragmentsof the Human genome disclosed herein. Examples of such assays can befound in Chard, T, An Introduction to Radioimmunoassay and RelatedTechniques, Elsevier Science Publishers, Amsterdam, The Netherlands(1986); Bullock, G. R. et al., Techniques in Immunocytochemistry,Academic Press, Orlando, Fla. Vol. 1 (1982), Vol. 2 (1983), Vol. 3(1985); Tijssen, P., Practice and Theory of Enzyme Immunoassays:Laboratory Techniques in Biochemistry and Molecular Biology, ElsevierScience Publishers, Amsterdam, The Netherlands (1985).

The test samples of the present invention include cells, protein ormembrane extracts of cells. The test sample used in the above-describedmethod will vary based on the assay format, nature of the detectionmethod and the tissues, cells or extracts used as the sample to beassayed. Methods for preparing nucleic acid extracts or of cells arewell known in the art and can be readily be adapted in order to obtain asample that is compatible with the system utilized.

In another embodiment of the present invention, kits are provided whichcontain the necessary reagents to carry out the assays of the presentinvention.

Specifically, the invention provides a compartmentalized kit to receive,in close confinement, one or more containers which comprises: (a) afirst container comprising one of the nucleic acid molecules that canbind to a fragment of the Human genome disclosed herein; and (b) one ormore other containers comprising one or more of the following: washreagents, reagents capable of detecting presence of a bound nucleicacid.

In detail, a compartmentalized kit includes any kit in which reagentsare contained in separate containers. Such containers include smallglass containers, plastic containers, strips of plastic, glass or paper,or arraying material such as silica. Such containers allows one toefficiently transfer reagents from one compartment to anothercompartment such that the samples and reagents are notcross-contaminated, and the agents or solutions of each container can beadded in a quantitative fashion from one compartment to another. Suchcontainers will include a container which will accept the test sample, acontainer which contains the nucleic acid probe, containers whichcontain wash reagents (such as phosphate buffered saline, Tris-buffers,etc.), and containers which contain the reagents used to detect thebound probe. One skilled in the art will readily recognize that thepreviously unidentified nuclear hormone receptor gene of the presentinvention can be routinely identified using the sequence informationdisclosed herein can be readily incorporated into one of the establishedkit formats which are well known in the art, particularly expressionarrays.

Vectors/host Sells

The invention also provides vectors containing the nucleic acidmolecules described herein. The term “vector” refers to a vehicle,preferably a nucleic acid molecule, which can transport the nucleic acidmolecules. When the vector is a nucleic acid molecule, the nucleic acidmolecules are covalently linked to the vector nucleic acid. With thisaspect of the invention, the vector includes a plasmid, single or doublestranded phage, a single or double stranded RNA or DNA viral vector, orartificial chromosome, such as a BAC, PAC, YAC, OR MAC.

A vector can be maintained in the host cell as an extrachromosomalelement where it replicates and produces additional copies of thenucleic acid molecules. Alternatively, the vector may integrate into thehost cell genome and produce additional copies of the nucleic acidmolecules when the host cell replicates.

The invention provides vectors for the maintenance (cloning vectors) orvectors for expression (expression vectors) of the nucleic acidmolecules. The vectors can function in prokaryotic or eukaryotic cellsor in both (shuttle vectors).

Expression vectors contain cis-acting regulatory regions that areoperably linked in the vector to the nucleic acid molecules such thattranscription of the nucleic acid molecules is allowed in a host cell.The nucleic acid molecules can be introduced into the host cell with aseparate nucleic acid molecule capable of affecting transcription. Thus,the second nucleic acid molecule may provide a trans-acting factorinteracting with the cis-regulatory control region to allowtranscription of the nucleic acid molecules from the vector.Alternatively, a trans-acting factor may be supplied by the host cell.Finally, a trans-acting factor can be produced from the vector itself.It is understood, however, that in some embodiments, transcriptionand/or translation of the nucleic acid molecules can occur in acell-free system.

The regulatory sequence to which the nucleic acid molecules describedherein can be operably linked include promoters for directing mRNAtranscription. These include, but are not limited to, the left promoterfrom bacteriophage λ, the lac, TRP, and TAC promoters from E. coli, theearly and late promoters from SV40, the CMV immediate early promoter,the adenovirus early and late promoters, and retrovirus long-terminalrepeats.

In addition to control regions that promote transcription, expressionvectors may also include regions that modulate transcription, such asrepressor binding sites and enhancers. Examples include the SV40enhancer, the cytomegalovirus immediate early enhancer, polyomaenhancer, adenovirus enhancers, and retrovirus LTR enhancers.

In addition to containing sites for transcription initiation andcontrol, expression vectors can also contain sequences necessary fortranscription termination and, in the transcribed region a ribosomebinding site for translation. Other regulatory control elements forexpression include initiation and termination codons as well aspolyadenylation signals. The person of ordinary skill in the art wouldbe aware of the numerous regulatory sequences that are useful inexpression vectors. Such regulatory sequences are described, forexample, in Sambrook et al., Molecular Cloning: A Laboratory Manual. 2nded., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,(1989).

A variety of expression vectors can be used to express a nucleic acidmolecule. Such vectors include chromosomal, episomal, and virus-derivedvectors, for example vectors derived from bacterial plasmids, frombacteriophage, from yeast episomes, from yeast chromosomal elements,including yeast artificial chromosomes, from viruses such asbaculoviruses, papovaviruses such as SV40, Vaccinia viruses,adenoviruses, poxviruses, pseudorabies viruses, and retroviruses.Vectors may also be derived from combinations of these sources such asthose derived from plasmid and bacteriophage genetic elements, e.g.cosmids and phagemids. Appropriate cloning and expression vectors forprokaryotic and eukaryotic hosts are described in Sambrook et al.,Molecular Cloning: A Laboratory Manual. 2nd. ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., (1989).

The regulatory sequence may provide constitutive expression in one ormore host cells (i.e. tissue specific) or may provide for inducibleexpression in one or more cell types such as by temperature, nutrientadditive, or exogenous factor such as a hormone or other ligand. Avariety of vectors providing for constitutive and inducible expressionin prokaryotic and eukaryotic hosts are well known to those of ordinaryskill in the art.

The nucleic acid molecules can be inserted into the vector nucleic acidby well-known methodology. Generally, the DNA sequence that willultimately be expressed is joined to an expression vector by cleavingthe DNA sequence and the expression vector with one or more restrictionenzymes and then ligating the fragments together. Procedures forrestriction enzyme digestion and ligation are well known to those ofordinary skill in the art.

The vector containing the appropriate nucleic acid molecule can beintroduced into an appropriate host cell for propagation or expressionusing well-known techniques. Bacterial cells include, but are notlimited to, E. coli, Streptomyces, and Salmonella typhimurium.Eukaryotic cells include, but are not limited to, yeast, insect cellssuch as Drosophila, animal cells such as COS and CHO cells, and plantcells.

As described herein, it may be desirable to express the peptide as afusion protein. Accordingly, the invention provides fusion vectors thatallow for the production of the peptides. Fusion vectors can increasethe expression of a recombinant protein, increase the solubility of therecombinant protein, and aid in the purification of the protein byacting for example as a ligand for affinity purification. A proteolyticcleavage site may be introduced at the junction of the fusion moiety sothat the desired peptide can ultimately be separated from the fusionmoiety. Proteolytic enzymes include, but are not limited to, factor Xa,thrombin, and enteronuclear hormone receptor. Typical fusion expressionvectors include pGEX (Smith et al., Gene 67:31-40 (1988)), pMAL (NewEngland Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.)which fuse glutathione S-transferase (GST), maltose E binding protein,or protein A, respectively, to the target recombinant protein. Examplesof suitable inducible non-fusion E. coli expression vectors include pTrc(Amann et al., Gene 69:301-315 (1988)) and pET 11d (Studier et al., GeneExpression Technology: Methods in Enzymology 185:60-89 (1990)).

Recombinant protein expression can be maximized in host bacteria byproviding a genetic background wherein the host cell has an impairedcapacity to proteolytically cleave the recombinant protein. (Gottesman,S., Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990)119-128). Alternatively, the sequence ofthe nucleic acid molecule of interest can be altered to providepreferential codon usage for a specific host cell, for example E. coli.(Wada et al., Nucleic Acids Res. 20:2111-2118 (1992)).

The nucleic acid molecules can also be expressed by expression vectorsthat are operative in yeast. Examples of vectors for expression in yeaste.g., S. cerevisiae include pYepSec1 (Baldari, et al., EMBO J. 6:229-234(1987)), pMFa (Kurjan et al., Cell 30:933-943(1982)), pJRY88 (Schultz etal., Gene 54:113-123 (1987)), and pYES2 (Invitrogen Corporation, SanDiego, Calif.).

The nucleic acid molecules can also be expressed in insect cells using,for example, baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., Sf9cells) include the pAc series (Smith et al., Mol. Cell Biol. 3:2156-2165(1983)) and the pVL series (Lucklow et al., Virology 170:31-39 (1989)).

In certain embodiments of the invention, the nucleic acid moleculesdescribed herein are expressed in mammalian cells using mammalianexpression vectors. Examples of mammalian expression vectors includepCDM8 (Seed, B. Nature 329:840(1987)) and pMT2PC (Kaufman et al., EMBOJ. 6:187-195 (1987)).

The expression vectors listed herein are provided by way of example onlyof the well-known vectors available to those of ordinary skill in theart that would be useful to express the nucleic acid molecules. Theperson of ordinary skill in the art would be aware of other vectorssuitable for maintenance propagation or expression of the nucleic acidmolecules described herein. These are found for example in Sambrook, J.,Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual.2nd, ed, Cold Spring Harbor Laboratory, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989.

The invention also encompasses vectors in which the nucleic acidsequences described herein are cloned into the vector in reverseorientation, but operably linked to a regulatory sequence that permitstranscription of antisense RNA. Thus, an antisense transcript can beproduced to all, or to a portion, of the nucleic acid molecule sequencesdescribed herein, including both coding and non-coding regions.Expression of this antisense RNA is subject to each of the parametersdescribed above in relation to expression of the sense RNA (regulatorysequences, constitutive or inducible expression, tissue-specificexpression).

The invention also relates to recombinant host cells containing thevectors described herein. Host cells therefore include prokaryoticcells, lower eukaryotic cells such as yeast, other eukaryotic cells suchas insect cells, and higher eukaryotic cells such as mammalian cells.

The recombinant host cells are prepared by introducing the vectorconstructs described herein into the cells by techniques readilyavailable to the person of ordinary skill in the art. These include, butare not limited to, calcium phosphate transfection,DEAE-dextran-mediated transfection, cationic lipid-mediatedtransfection, electroporation, transduction, infection, lipofection, andother techniques such as those found in Sambrook, et al. (MolecularCloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

Host cells can contain more than one vector. Thus, different nucleotidesequences can be introduced on different vectors of the same cell.Similarly, the nucleic acid molecules can be introduced either alone orwith other nucleic acid molecules that are not related to the nucleicacid molecules such as those providing trans-acting factors forexpression vectors. When more than one vector is introduced into a cell,the vectors can be introduced independently, co-introduced or joined tothe nucleic acid molecule vector.

In the case of bacteriophage and viral vectors, these can be introducedinto cells as packaged or encapsulated virus by standard procedures forinfection and transduction. Viral vectors can be replication-competentor replication-defective. In the case in which viral replication isdefective, replication will occur in host cells providing functions thatcomplement the defects.

Vectors generally include selectable markers that enable the selectionof the subpopulation of cells that contain the recombinant vectorconstructs. The marker can be contained in the same vector that containsthe nucleic acid molecules described herein or may be on a separatevector. Markers include tetracycline or ampicillin-resistance genes forprokaryotic host cells and dihydrofolate reductase or neomycinresistance for eukaryotic host cells. However, any marker that providesselection for a phenotypic trait will be effective.

While the mature proteins can be produced in bacteria, yeast, mammaliancells, and other cells under the control of the appropriate regulatorysequences, cell-free transcription and translation systems can also beused to produce these proteins using RNA derived from the DNA constructsdescribed herein.

Where secretion of the peptide is desired, which is difficult to achievewith multi-transmembrane domain containing proteins such as nuclearhormone receptors, appropriate secretion signals are incorporated intothe vector. The signal sequence can be endogenous to the peptides orheterologous to these peptides.

Where the peptide is not secreted into the medium, which is typicallythe case with nuclear hormone receptors, the protein can be isolatedfrom the host cell by standard disruption procedures, including freezethaw, sonication, mechanical disruption, use of lysing agents and thelike. The peptide can then be recovered and purified by well-knownpurification methods including ammonium sulfate precipitation, acidextraction, anion or cationic exchange chromatography, phosphocellulosechromatography, hydrophobic-interaction chromatography, affinitychromatography, hydroxylapatite chromatography, lectin chromatography,or high performance liquid chromatography.

It is also understood that depending upon the host cell in recombinantproduction of the peptides described herein, the peptides can havevarious glycosylation patterns, depending upon the cell, or maybenon-glycosylated as when produced in bacteria. In addition, the peptidesmay include an initial modified methionine in some cases as a result ofa host-mediated process.

Uses of Vectors and Host Cells

The recombinant host cells expressing the peptides described herein havea variety of uses. First, the cells are useful for producing a nuclearhormone receptor protein or peptide that can be further purified toproduce desired amounts of nuclear hormone receptor protein orfragments. Thus, host cells containing expression vectors are useful forpeptide production.

Host cells are also useful for conducting cell-based assays involvingthe nuclear hormone receptor protein or nuclear hormone receptor proteinfragments, such as those described above as well as other formats knownin the art. Thus, a recombinant host cell expressing a native nuclearhormone receptor protein is useful for assaying compounds that stimulateor inhibit nuclear hormone receptor protein function.

Host cells are also useful for identifying nuclear hormone receptorprotein mutants in which these functions are affected. If the mutantsnaturally occur and give rise to a pathology, host cells containing themutations are useful to assay compounds that have a desired effect onthe mutant nuclear hormone receptor protein (for example, stimulating orinhibiting function) which may not be indicated by their effect on thenative nuclear hormone receptor protein.

Genetically engineered host cells can be further used to producenon-human transgenic animals. A transgenic animal is preferably amammal, for example a rodent, such as a rat or mouse, in which one ormore of the cells of the animal include a transgene. A transgene isexogenous DNA which is integrated into the genome of a cell from which atransgenic animal develops and which remains in the genome of the matureanimal in one or more cell types or tissues of the transgenic animal.These animals are useful for studying the function of a nuclear hormonereceptor protein and identifying and evaluating modulators of nuclearhormone receptor protein activity. Other examples of transgenic animalsinclude non-human primates, sheep, dogs, cows, goats, chickens, andamphibians.

A transgenic animal can be produced by introducing nucleic acid into themale pronuclei of a fertilized oocyte, e.g., by microinjection,retroviral infection, and allowing the oocyte to develop in apseudopregnant female foster animal. Any of the nuclear hormone receptorprotein nucleotide sequences can be introduced as a transgene into thegenome of a non-human animal, such as a mouse.

Any of the regulatory or other sequences useful in expression vectorscan form part of the transgenic sequence. This includes intronicsequences and polyadenylation signals, if not already included. Atissue-specific regulatory sequence(s) can be operably linked to thetransgene to direct expression of the nuclear hormone receptor proteinto particular cells.

Methods for generating trasgenic animals via embryo manipulation andmicroinjection, particularly animals such as mice, have becomeconventional in the art and are described, for example, in U.S. Pat.Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No.4,873,191 by Wagner et al. and in Hogan, B., Manipulating the MouseEmbryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1986). Similar methods are used for production of other transgenicanimals. A transgenic founder animal can be identified based upon thepresence of the transgene in its genome and/or expression of transgenicmRNA in tissues or cells of the animals. A transgenic founder animal canthen be used to breed additional animals carrying the transgene.Moreover, transgenic animals carrying a transgene can further be bred toother transgenic animals carrying other transgenes. A transgenic animalalso includes animals in which the entire animal or tissues in theanimal have been produced using the homologously recombinant host cellsdescribed herein.

In another embodiment, transgenic non-human animals can be producedwhich contain selected systems that allow for regulated expression ofthe transgene. One example of such a system is the cre/loxP recombinasesystem of bacteriophage P1. For a description of the cre/loxPrecombinase system, see, e.g., Lakso et al. PNAS 89:6232-6236 (1992).Another example of a recombinase system is the FLP recombinase system ofS. cerevisiae (O'Gorman et al. Science 251:1351-1355 (1991). If acre/loxP recombinase system is used to regulate expression of thetransgene, animals containing transgenes encoding both the Crerecombinase and a selected protein is required. Such animals can beprovided through the construction of “double” transgenic animals, e.g.,by mating two transgenic animals, one containing a transgene encoding aselected protein and the other containing a transgene encoding arecombinase.

Clones of the non-human transgenic animals described herein can also beproduced according to the methods described in Wilmut, I. et al. Nature385:810-813 (1997) and PCT International Publication Nos. WO 97/07668and WO 97/07669. In brief, a cell, e.g., a somatic cell, from thetransgenic animal can be isolated and induced to exit the growth cycleand enter G_(o) phase. The quiescent cell can then be fused, e.g.,through the use of electrical pulses, to an enucleated oocyte from ananimal of the same species from which the quiescent cell is isolated.The reconstructed oocyte is then cultured such that it develops tomorula or blastocyst and then transferred to pseudopregnant femalefoster animal. The offspring born of this female foster animal will be aclone of the animal from which the cell, e.g., the somatic cell, isisolated.

Transgenic animals containing recombinant cells that express thepeptides described herein are useful to conduct the assays describedherein in an in vivo context. Accordingly, the various physiologicalfactors that are present in vivo and that could effect substratebinding, nuclear hormone receptor protein activation, and signaltransduction, may not be evident from in vitro cell-free or cell-basedassays. Accordingly, it is useful to provide non-human transgenicanimals to assay in vivo nuclear hormone receptor protein function,including substrate interaction, the effect of specific mutant nuclearhormone receptor proteins on nuclear hormone receptor protein functionand substrate interaction, and the effect of chimeric nuclear hormonereceptor proteins. It is also possible to assess the effect of nullmutations, that is mutations that substantially or completely eliminateone or more nuclear hormone receptor protein functions.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the above-described modesfor carrying out the invention which are obvious to those skilled in thefield of molecular biology or related fields are intended to be withinthe scope of the following claims.

4 1 2086 DNA Homo sapiens 1 aacagcacga gggcgagggg acgtctcctc tcccccagctgctctgctcg gatggcgccg 60 ccggctgagt gacgggggcg gcgcgcagga cttcccagctcggacctctt gccttcgagg 120 ggaaagatgt acgagagtgt agaagtgggg ggtcccacccctaatccctt cctagtggtg 180 gatttttata accagaaccg ggcctgtttg ctcccagagaaggggctccc cgccccgggt 240 ccgtactcca ccccgctccg gactccgctt tggaatggctcaaaccactc cattgagacc 300 cagagcagca gttctgaaga gatagtgccc agccctccctcgccaccccc tctaccccgc 360 atctacaagc cttgctttgt ctgtcaggac aagtcctcaggctaccacta tggggtcagc 420 gcctgtgagg gctgcaaggg cttcttccgc cgcagcatccagaagaacat ggtgtacacg 480 tgtcaccggg acaagaactg catcatcaac aaggtgacccggaacccctg ccagtactgc 540 cgactgcaga agtgctttga agtgggcatg tccaaggagtctgtgagaaa cgaccgaaac 600 aagaagaaga aggaggtgcc caagcccgag tgctctgagagctacacgct gacgccggag 660 gtgggggagc tcattgagaa ggtgcgcaaa gcgcaccaggaaaccttccc tgccctctgc 720 cagctgggca aatacactac gaacaacagc tcagaacaacgtgtctctct ggacattgac 780 ctctgggaca agttcagtga actctccacc aagtgcatcattaagactgt ggagttcgcc 840 aagcagctgc ccggcttcac caccctcacc atcgccgaccagatcaccct cctcaaggct 900 gcctgcctgg acatcctgat cctgcggatc tgcacgcggtacacgcccga gcaggacacc 960 atgaccttct cggacgggct gaccctgaac cggacccagatgcacaacgc tggcttcggc 1020 cccctcaccg acctggtctt tgccttcgcc aaccagctgctgcccctgga gatggatgat 1080 gcggagacgg ggctgctcag cgccatctgc ctcatctgcggagaccgcca ggacctggag 1140 cagccggacc gggtggacat gctgcaggag ccgctgctggaggcgctaaa ggtctacgtg 1200 cggaagcgga ggcccagccg cccccacatg ttccccaagatgctaatgaa gattactgac 1260 ctgcgaagca tcagcgccaa gggggctgag cgggtgatcacgctgaagat ggagatcccg 1320 ggctccatgc cgcctctcat ccaggaaatg ttggagaactcagagggcct ggacactctg 1380 agcggacagc cggggggtgg ggggcgggac gggggtggcctgcccccccc gccaggcagc 1440 tgtagcccca gcctcagccc cagctccaac agaagcagcccggccaccca ctccccgtga 1500 ccgcccacgc cacatggaca cagccctcgc cctccgccccggcttttctc tgcctttcta 1560 ccgaccatgt gaccccgcac cagccctgcc cccacctgccctcccgggca gtactgggga 1620 ccttccctgg gggacgggga gggaggaggc agcgactccttggacagagg cctgggccct 1680 cagtggactg cctgctccca cagcctgggc tgacgtcagaggccgaggcc aggaactgag 1740 tgaggcccct ggtcctgggt ctcaggatgg gtcctgggggcctcgtgttc atcaagacac 1800 ccctctgccc agctcaccac atcttcatca ccagcaaacgccaggacttg gctcccccat 1860 cctcagaact cacaagccat tgctccccag ctggggaacctcaacctccc ccctgcctcg 1920 gttggtgaca gagggggtgg gacaggggcg gggggttccccctgtacata ccctgccata 1980 ccaaccccag gtattaattc tcgctggttt tgtttttattttaatttttt tgttttgatt 2040 tttttaataa gaattttcat tttaagcaca aaaaaaaaaaaaaaaa 2086 2 457 PRT Homo sapiens 2 Met Tyr Glu Ser Val Glu Val Gly GlyPro Thr Pro Asn Pro Phe Leu 1 5 10 15 Val Val Asp Phe Tyr Asn Gln AsnArg Ala Cys Leu Leu Pro Glu Lys 20 25 30 Gly Leu Pro Ala Pro Gly Pro TyrSer Thr Pro Leu Arg Thr Pro Leu 35 40 45 Trp Asn Gly Ser Asn His Ser IleGlu Thr Gln Ser Ser Ser Ser Glu 50 55 60 Glu Ile Val Pro Ser Pro Pro SerPro Pro Pro Leu Pro Arg Ile Tyr 65 70 75 80 Lys Pro Cys Phe Val Cys GlnAsp Lys Ser Ser Gly Tyr His Tyr Gly 85 90 95 Val Ser Ala Cys Glu Gly CysLys Gly Phe Phe Arg Arg Ser Ile Gln 100 105 110 Lys Asn Met Val Tyr ThrCys His Arg Asp Lys Asn Cys Ile Ile Asn 115 120 125 Lys Val Thr Arg AsnPro Cys Gln Tyr Cys Arg Leu Gln Lys Cys Phe 130 135 140 Glu Val Gly MetSer Lys Glu Ser Val Arg Asn Asp Arg Asn Lys Lys 145 150 155 160 Lys LysGlu Val Pro Lys Pro Glu Cys Ser Glu Ser Tyr Thr Leu Thr 165 170 175 ProGlu Val Gly Glu Leu Ile Glu Lys Val Arg Lys Ala His Gln Glu 180 185 190Thr Phe Pro Ala Leu Cys Gln Leu Gly Lys Tyr Thr Thr Asn Asn Ser 195 200205 Ser Glu Gln Arg Val Ser Leu Asp Ile Asp Leu Trp Asp Lys Phe Ser 210215 220 Glu Leu Ser Thr Lys Cys Ile Ile Lys Thr Val Glu Phe Ala Lys Gln225 230 235 240 Leu Pro Gly Phe Thr Thr Leu Thr Ile Ala Asp Gln Ile ThrLeu Leu 245 250 255 Lys Ala Ala Cys Leu Asp Ile Leu Ile Leu Arg Ile CysThr Arg Tyr 260 265 270 Thr Pro Glu Gln Asp Thr Met Thr Phe Ser Asp GlyLeu Thr Leu Asn 275 280 285 Arg Thr Gln Met His Asn Ala Gly Phe Gly ProLeu Thr Asp Leu Val 290 295 300 Phe Ala Phe Ala Asn Gln Leu Leu Pro LeuGlu Met Asp Asp Ala Glu 305 310 315 320 Thr Gly Leu Leu Ser Ala Ile CysLeu Ile Cys Gly Asp Arg Gln Asp 325 330 335 Leu Glu Gln Pro Asp Arg ValAsp Met Leu Gln Glu Pro Leu Leu Glu 340 345 350 Ala Leu Lys Val Tyr ValArg Lys Arg Arg Pro Ser Arg Pro His Met 355 360 365 Phe Pro Lys Met LeuMet Lys Ile Thr Asp Leu Arg Ser Ile Ser Ala 370 375 380 Lys Gly Ala GluArg Val Ile Thr Leu Lys Met Glu Ile Pro Gly Ser 385 390 395 400 Met ProPro Leu Ile Gln Glu Met Leu Glu Asn Ser Glu Gly Leu Asp 405 410 415 ThrLeu Ser Gly Gln Pro Gly Gly Gly Gly Arg Asp Gly Gly Gly Leu 420 425 430Pro Pro Pro Pro Gly Ser Cys Ser Pro Ser Leu Ser Pro Ser Ser Asn 435 440445 Arg Ser Ser Pro Ala Thr His Ser Pro 450 455 3 17000 DNA Homo sapiensmisc_feature (1)...(17000) n = A,T,C or G 3 gtccttgggt agcatgtacatttccatccc ttccttttat atatgggggt aataggatac 60 cccctcctcc aggggtatcccctctttcta gggacctacc caagctaggc ctttcttcca 120 gtgaaacgtg catcccgagggcttctagga tgaagtagtc cactggaagg caccagctct 180 tccttttatc tctccagagctggacagtgc accaggggcc ggtactggtt ccccagctag 240 gagacacctt gggcggggctttgctcgccg gaagcacgca gagcgtgggg aggagggccc 300 cctctgcctg tgtttgtgccaacagcaccc gcgctgccgc gtcgggttcc ggcggccgga 360 gtcacacatg atgtcacagacaatgacaca agccggtgtc tcattccgac acagcgtccg 420 agctgcacaa tgtcacacccgggtgccaaa cacttggccc cgcgcgaccc ggccctacgc 480 ctcctgccgc cgctctccgcgtctccgggg gaggtggccc ggttcggccg ggcagggggc 540 tggcgggcga gccccgcgggcgggctggcg agcgggtgat gtcacgggca gcggtgggtg 600 ggtcactcgg aggtgaggcgccgccaggcg agttcagcga gagttcagcc gcattgcatt 660 aggcaaatga ggcccggcctgggtgggggt gtgtgttaag gggaggacac cgggaccacc 720 cccctcttcc ccgccccaccacctcctcca ccacggcttc gctcggccag ggactgacca 780 aaccttgggg gagcctgggagccggaactg gtacaagggg aggacgcccg cccctcttcc 840 gtccttgtcc cctcgcagccccctcctctc cctgtactcg gcgtccctct gtactctgtg 900 tactcctcat ctggagcctttcccccttcc tgcttctctc ctctcctccc ccttcccagg 960 ctgcccccac ttgcctgtccacatgccgcc tctccctctc ggttccctgc gtttctcccg 1020 ctgcagccgg acgcgccgggaatgggttaa gccaggggcg gtgcctggac ggggcggggc 1080 ggtggaaagg gggtggtgcccggaggggag ggggcgcgca gagctggggt gggggggccg 1140 tggcgcgtac caccagagaccgagcgagtc gccagctgcc cctggcctgg cgggggcgga 1200 accgcgcggg atccccacccccacccggaa tcctcgccac ggagaatccc tggagaagcc 1260 ccggatcccc ggctgggaggaggaagtgct cgttgacccc cagccccgcg ctgatcccgc 1320 ccccggcctg cggacttggggagccgctgt actctgcctc ggacgccacg agactctaga 1380 cgggagtccc ctcgaggtgaagccgctgag ttcccgggcc ccgccaggct tccctgggag 1440 agccgacgga ccccccctcccagcacacac aacttccctg cttttcaccg ggactggcgg 1500 agcggccggc ggacttagacgcggggactt cagggcaggg ggcgccccct gcccgggtca 1560 ccagtcgggg cgaggggacgtctcctctcc cccagctgct ctgctcggat ggcgccgccg 1620 gctgagtgac gggggcggcgcgcaggactt cccagctcgg acctcttgcc ttcgagggga 1680 aagatgtacg agagtgtagaagtggggggt cccaccccta atcccttcct agtggtggat 1740 ttttataacc agaaccgggcctgtttgctc ccagagaagg ggctccccgc cccgggtccg 1800 tactccaccc cgctccggactccgctttgg aatggctcaa accactgtac gtaccggcct 1860 ctcagtctgc tgttgtagggggtgggagtg ggcggtaggg cttccactac tactcggggg 1920 tgagagtccc ggggtgtagtggaggtcctg tctctacctt tcacttaacc cgtgttgccc 1980 ttgctggaca attgaaccctcccggccgca ccctcccccc agtaacccta agtgcaattt 2040 gtgttagatt agggctgaggaactttgaga gttccttctt ttcaagcaac attcctttca 2100 tctctttgtt tcacttcttcccaggagaaa tgaagcccaa gccccctttg gcccccagtt 2160 tgtatattct ttcttggccttgggaaatcc caaaaaggtt tcaccagcaa ggcttgggaa 2220 ggggtggggg ggtaaaagggttccctggtc ttgtggtggg tttttggtct tgcttacccg 2280 ggggggnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnngaag 2340 ggctctgtgc acactcaggagctcggagca ccagggtgta cacctgggca ttttcctgcg 2400 cagctgtgag gcagtgtacactgggtgggc gggagcaggc gcaagggggt tattgttaga 2460 tggctcaggt ttcttcccctcctgggcttt gggctctttg ctggagggga agctcttccg 2520 tggaggatct cccaccttcctggacctgct gcctccctcc tgcctgccag ggaggagggg 2580 tggagtgggt ctcgggggggccctggcaga ttggaaaagg ttgaagggca aaggacttac 2640 cccacccctc ttgctgggagaagagagacc tgagatggac agacagccca cctctgccct 2700 cccagagcca cttctatcccagcttttcct attgtcctgc ccccgaccat ttcctctagg 2760 gccgaatctg ctgtgtggctgtagacacaa gagggaaggt atcacccttg actttggaag 2820 aagagagagt gagaggatgactctaggacc ctttttctca ttctcccagt gctggagcaa 2880 gacccccctc ccctagggggatagttggag cagggctgcc cagagtcacc ccttccactg 2940 ccttggccac cttctccagagggctggaga gaagctggga tctgagacct tggtctccag 3000 cccctgtctc ttcttagcccatggggacag ctcagctctt cctggcccag aactggagag 3060 ggaggaggat cacagagagtaggacaggca gtgtattggt gagcccttcc cctaaaccac 3120 tggacatggg gaagtggagacctgtcccca catccattct ggggtggggt agtagaccta 3180 gaggcctggg tttccagttcccgtagtctg agcgtgggtg tgcatatata agtgagtgag 3240 gtgtcagtgg actcgggtcctgaggctgtg aggttgggag tgatgggggt ctgggggctt 3300 gccttgaggc acaggaaggacccggagtct gagggtggca actagactca gtctagaata 3360 tgtggggcca atgccaccaccttggaaggg tccccttggg tgtgttggaa gtccgctggt 3420 gactggagct gcctccagccccctcttggg gaattctcca ctctcccctt tactgccact 3480 gaaggtggga agagcaggttggctctggga ggaggtggcc tgggttctgc agggccctag 3540 ggacattgcc tccctccccagagccctcat ttcggtgcat tagaggacaa gggggggtgc 3600 acaggatgtg gctccccatctgtctcccac caatctccgc cactcacacc tccgcccgct 3660 cccagacgtc caagaatgtgaagcacgtgg atgcccgtag ttgggggagg gggagacgct 3720 tatcaggcgg ccgctgggctaggggccttc ttccgctgcc gcggtacacc cagagctacc 3780 cccgcctctc cccgggaggaggaaggacgg tacagagggc cctacgcccc ctccccaacc 3840 atccccaggg gctgcgaggggagctgcgga ggagcgggcg ccagctggat tgggagggga 3900 gccgctggcc gggggcccggctgatttcct gctgatctcc tccaggaaac cggccccttg 3960 tgcgagcctg cgaacggctcgggggcgtgg ggaatccgga gtggagcgct ctgcgccgcc 4020 cgccctgcca ggatggggagcgagggaggg gcaccctggc agcgtcggcg ggaggggacg 4080 cctggcttcc tgggtcagttccagtcctct gttgggcgct ggaactttga gctgagaagg 4140 tgtggtcctt ctctagcccgagtccttctg caggaagagg agagattggt gggctgggcc 4200 tctggggagg gaggttagcagggatgggcc aggcccgggc agtccctccc ccgttggtgt 4260 ccctccccac tccacctgtgtgtgcaggga gttatggccg tgtcctaact cttgcagagg 4320 ctgtgaggat tccggagttccccacacctc cggccttggt ccttgtacct cacctccttg 4380 gactgctggc tggaggcctggggaggtggg gcatcgagct ctgggttcaa agggcagagc 4440 agggaaacct cagagctgggttacctgggt gacaggtggg gatgtgctgg aggtaggggg 4500 caggctatgt tacagcctccaaggcagtca agctgccgtt gggtgggcta aaaggaggcc 4560 ttgcccagcc taaactgtagtccttgcctc tggtcatctc tcccattctg ccaaaaaata 4620 attttaaaaa gcacattctctcagttccgt aaacaccctc tgttggactt tgctttagct 4680 ccatgttttt atggctttttgccctctagt ctgtcccagg ccttagagct gtttacctct 4740 catcctggta tcccccatgactccccatac cctagctccc ctcgtgacat cccgctctgt 4800 acccccaaag ctccctcagtcctttctccc tctccagtct ggttcatttt agaagtgggg 4860 ccttgggaga ggcggggcccagggcaaacg gtggattagg aggggtgggg aggtcagtgc 4920 cttcttcctc tgcttgtcggaatgctgacc aagattctag gccatggtcc ccccaaccct 4980 ccacataccc ccttgcccttgatctcccct ccccccacca gtctggattg tctattgtta 5040 ctgcttttac gtcttggaaaaagttagcac aacaaagggc tgctttgtgg ctcaccccct 5100 ctgcctcctg gcctcacccaggccccccaa ccccgccccc ccagcagctg ttctcaggcc 5160 tctcagcctg tctgatttgcttgtctggcc tggggagaat gaggtgggag aaaaccaggc 5220 cagggcagtt ggtgttggagtgaagagcag acggcggtgg ggaggtcagg agagaatctg 5280 ctgggctggg gatggtgtgggcatcaactg tcccattgct gcaggctggt cttggggcag 5340 ggaaggggat ggggggccatagcagtgctg gtcagccagg ctggcctggg aagtggtgcc 5400 caggcactac taagagccaggaaagccctg ccaaggttgt tggcctagtt ccctgtcatc 5460 agccgcctag cagcccccactgtgtctgca ggtaaggggg gagggtggta gcacatagtc 5520 agcccctggt gttcccatgcttccttcctc tgtgccccaa ttttagggcc atgtgatttg 5580 gggctatgtg actcatgtctgtaaggtgct tgggccagga gctgtgggca cctttaaatg 5640 ccagccagtc tcatgtgccggagtttgggg tagggctagg taggattgtg gaatatggga 5700 ggaggcaggg atctgtctacctagggaggc atcctcatcc atccttggcc ctggacaaga 5760 gaacttgaac gttggtaggggcctcaggac gatgctgcgt ggccccttgg gaatctggga 5820 ttgtcctggt catagttcttatcttgcacc caacaccctt agctgcccag gctttggaca 5880 tggatagccc ctacccaacccagccctgtt ctgcctacag tgatgggcat ggagccagac 5940 actggggagg atttggccagtgagggctgc ccctgctgtc tgggtcaccc ctcctggctg 6000 ccctcttgga gctgaataacagaaggggag gggttagtaa cccggacata gtattgaggc 6060 cagacagaca gagcattgatgggaacagac cccctttgtc atgccatctc tccccagatg 6120 gggggtaccc agaataatgggcttttgggg ccctggggac tcttctccct gtattcaggg 6180 tatctccccc tatctcagggagacacctcc tactgtgccc agcatttgtg actcttcttt 6240 gcaccccctg ccttgggtccctggccctgg gattgtttgg gtggaggagg ggcagtggct 6300 gctggcagaa tggggtggaggggggagcgg aagcagaggg ggcgggggag tggccggctt 6360 tgaatatcct gttgaccccagtttcctctg cccccagctt atgtcctctt ccctccctcc 6420 tcttcaagcg ttaactccttcctaactcgg ggggagaacg gggccaggcc gcccaggggc 6480 aggagcttta gaatcagggtgacccccacc cctactcccc aagcacagtc acggcacaca 6540 tacaaatgtg atggtttatcattgtatctt tgtggttttg aaggtggggg tcctaggagt 6600 ccagaggagt gatggggtgctggaggcttc attggcagcc tcctgccctg agtctggctg 6660 gggagtccca gttttcttaagacttgaatc ctgccagcag tggtgaggct gggagaggct 6720 cttaggaggg acggtgaggcagggtggagc ttggtactaa ggatggcgac ctaggtctct 6780 aactgcccct cccctcttctctctctagcc attgagaccc agagcagcag ttctgaagag 6840 atagtgccca gccctccctcgccaccccct ctaccccgca tctacaagcc ttgctttgtc 6900 tgtcaggaca agtcctcaggctaccactat ggggtcagcg cctgtgaggg ctgcaaggtg 6960 agttgaaggg gtcattgggaaggacagctt gatgaggtca atggggatgt ccccacttct 7020 gtgtcctggg agtgtgcagttggggggtgt ccctgaattg ctgctcttct ttctctgtgg 7080 aagttggcag caagcaggggacacctacca cagtttcccc acaggtcctc ccccataaat 7140 gtgcagggct ccctcaaaccagaggtcccc tcctgcctca gctcctttcc ctgtctctat 7200 cctccagctg gcagggcgtacgcctgctct gccaccgctg cccaggttgc catggtgagc 7260 tggctgccga ctggctcttggctggggacc caggaggcct cccccggcgg ccctgcctga 7320 acctcaccat ggcagcctggcaggaggcag ttaggagcag gcaccctgcc ttagcttccc 7380 cttcaggtgc ccgggctgtgggctccccag tgtctggctg gatttcccca tcctcacgtt 7440 aggtgccagg gtgcaggtatacctggtcct tagcagccct gcgcccggct tctcctcctt 7500 tccctggggc ctgagcctctgtgtgcgttt cttcctccag agattggggc tcagaatctt 7560 cacagctttg ggccttgcagctctgggctg ctcttcagcc tggagtagct atccccagat 7620 gtgggacgga ggtcaagggcaaagcacaag gactcaggct gtgtgtctgc ctgtcctgtc 7680 tggttgttcc tggtctgttcttcctctgtc cgcctgtccc tctggtcagc ctgtatgtgg 7740 agcccctggc cagcctgggtctgtgtctgt gatgggtcgg tgcacacctg tcttggtgaa 7800 ctcacatctt tctgccttgctcctgagtgc atgtgtgtgt tcgcctccat ttctctggcc 7860 agcccgtgta tctgcctcctggcctcttcg ggcttgtctt cttttcctgt gttctgagtt 7920 caggggtgtg ggttccagatccctggctgt tgcccagtta gccccatgtc ttcctatttc 7980 tgactcacca gcagccctgaggtcttttcc ctggaaggga ggagtcaggt gtgtgctgtg 8040 ggttggggga agactcctgcccatcctgca gtgttgaggc aggtactggg attctcctga 8100 ggaggatcct tttaggtgaatcattctccc cagcttttct ggcctgctca ggtaggcgat 8160 gggcaaacgc ttgggggcagcagctggcct ggccctcctc ccctagactg agaccgtagc 8220 caggcactgc tcccactgtgggtgtggaca acctgactcc ctcccctcca tacccagggc 8280 ttcttccgcc gcagcatccagaagaacatg gtgtacacgt gtcaccggga caagaactgc 8340 atcatcaaca aggtgacccggaaccgctgc cagtactgcc gactgcagaa gtgctttgaa 8400 gtgggcatgt ccaaggagtgtgagtgccat agggcagggg ccgagtcccg cctcagttgg 8460 ggtctcagat gctcctaaagaccaagggag cagggctctg tggatgtttg tgcacatgca 8520 tgaacacgca tgccgtggtgtgcgggctca cggttgagga tggtttgtgt gtagctgcaa 8580 ggacctgttt gcgagtctggctggctgtgt gtccacgggc aggtctgtgc tccgggaccg 8640 tgtatgtgta accattcctgtttctgcacg tctggctgtg tgtgcttgcg tatgtgtgtg 8700 tgtgtgcatg ctccaggatggctttcttcc aggccgtgct tggttttggg gtggggctca 8760 gaggcatagg cagtcccttctgattgtgag tcttagggga ggggcttgaa ttctgagggg 8820 tgcttggctg gacttatgtgtgtatggggg ggtggaaggg ctggcacaag gatccaaaag 8880 ccattgtcta gttaagcctgggattcagag ttggaagaaa gaattgggac ttctcagatc 8940 ccagaggaaa cggggtttccactttgggct cagctgaggc ctgatggagg gagggaggga 9000 aaggctggac agggagaccctcttgtgttg aatcatgggt gttgccatgg tgaccggtga 9060 ttgatgatgt cagagataaatgacgctgac agacgcctcc ttgtctgcgt ggccgttgcc 9120 atggagcctg agccttgggggatgggatgg gggagggggc tgcaggaccc cctagccctt 9180 tgtggggagg gcagtggggagggggcacgg gtgagatggt tctgactgtt gcacgaagag 9240 ccccagacag gaatggaggggactggagtg tcctgccaca ggaggctggg ggtgccttgt 9300 cctgagccca ggaagtggtggctcctgctg caagagtggg tgacaactca agacccacaa 9360 gcctggaacc cttcgcttaagggctgtcac ctcctcctct ctgtttgtgc caccttctgc 9420 tcttttcatg gcagaaggaccagggagggg accccttctc cctcccaccg ccaactcccc 9480 ttctccctcc caccgccaactccccctctc ccggctgctc tgtgccccgg agctgagcag 9540 ctgccatttc aatagaattaaagcttccga atgataaacg tcttgtcaca gctgcaattt 9600 tctcttccca aattatccccccactctccc tctccctctc ccttctctcc cctgcacttt 9660 attgaatttg cagaatcgacatgagtgatc tccaaattat gccagctacc cccacctcgc 9720 taccccctcc ctgagcccctcccccaccct cccttcctcc cgcgtcagca gccaccacca 9780 ccagccctgt gagtgattgtgtgtctggat aatcggctgg taacgacccc atcgcttctt 9840 taaagccgag tggtgtgtgcggctcagcgc ccctggtgat ttgtcagctc cccagctaat 9900 gggccaagag attctccccgccaggtcccc cactctcagg ctggggagcc ctactcccca 9960 cttgccccag gagctgctcagagccagtcc caagggaccc ccagggagac tgcagctggg 10020 agggctgggt gagtggaggcgggagaagga ccttcctggg gaaagaggag gcagagcacc 10080 taggagggca ccgtcgcctggagtgtgagc tggagtagac gcgtggggga tagcatgcgg 10140 ctggctatgg ggtggggtggggggtgtgtg cagggccaca gctgtgctca tggggcttct 10200 ggggcagaac ttgatgtgtgggttgggtgg gcatggaggg ctggagtgcg tggcaatgcc 10260 ttgcctgccc gtgaacgcgtgctgtgtgcg cgtgcttaca agcctgggtg acctcctcag 10320 cagctggcag ctctctgtcaggctgggggt ggacgaggcc ctgagcagcc tgcagctgcc 10380 ctcttaaccc cctctgccctccacagctgt gagaaacgac cgaaacaaga agaagaagga 10440 ggtgcccaag cccgagtgctctgagagcta cacgctgacg ccggaggtgg gggagctcat 10500 tgagaaggtg cgcaaagcgcaccaggaaac cttccctgcc ctctgccagc tgggcaaata 10560 cactacggta tggctttcccccggcctgca gggtgggatt tgcccagggc cacagggcca 10620 ggatgggccc ctctcaggcaccccttcttg tgccaggcaa gatctctgcg tccttccctt 10680 cccctctctt ctccctcctcctgctgcctc ttcccaagga gctcccagga agtgaaggct 10740 gggtagaggg caggcctgtgggggctggag ccaggctgag aaggggtgcc atggagaaga 10800 aggccctcac tctccctcctcccccagaac aacagctcag aacaacgtgt ctctctggac 10860 attgacctct gggacaagttcagtgaactc tccaccaagt gcatcattaa gactgtggag 10920 ttcgccaagc agctgcccggcttcaccacc ctcaccatcg ccgaccagat caccctcctc 10980 aaggctgcct gcctggacatcctggtgagg gtctgcaccc tggcccccag gcactgcccc 11040 tgtgtcctgg gtagatgtccttccagccag acagccaccc tcctaaatgt ctgtctgcaa 11100 tcaacctgtc caaatgcccaccgcccaaat gtctgccctt cctctcccca tatgtccacc 11160 tgtccactcg tctccctgtccactcagcca cctagcagcc agatgtgcag gagctcacct 11220 gttcacccat acacatatccagccacccag ccatccatcc atttagccag taataaagat 11280 tcacgtagga gccaggtgcagtggctcata cctgtaatcc cagcactttg ggaggccgag 11340 cgaggcagga ggatcacttgaggctggaag ttcaagacca ccctgggcaa catagtgaga 11400 ccttatttct gcaaaaaactaaaaagattc acctaggatc ctctggccag tgttcgagct 11460 gggtgtcagg aacccagcggtgaatgcacc accatcccct ctcttgaaaa ccttccatgt 11520 gaggcaagag ataagtcaacagaggttgca aaactgtgat caatgcttcc tggagattgg 11580 gggagggctt gtgactgcttgggcctgaag gatgatgtct cagaggaggt gacatctagg 11640 ggtttgtaga gggggaggtgagagggtagc cctaactcag gagcaggaag tgaaagactt 11700 gctgctgtga ggccatgctgagctcagggg actgccgggc actcggtgag gtgagcccga 11760 gggtagactg ggctggaggctggatgcagg gggtgggggc aggaagaggt ggtgggaact 11820 gccaaagcct aggctggagggagcactctc cttcctgctg tccctgacaa gggctcggtc 11880 cacctgttcc ctcttggtcacctccagggt ggggaacctg ggatttgacg agactgtcat 11940 ttctttttat gtttttcttttttgagatgg agtttcactc ttgtcaccca ggctggagtg 12000 cagtagtatg atcttggctcactgcagcct gcaactgctg cctcccgggt tcaagcgatt 12060 ctcctgcctc agcctcctgagtagctggga ttacaggcac ccgccaccac acccggctaa 12120 tttttgtatt tttgtagagacggggtttca ccatgttggc caggccggtc tcgaactcct 12180 gacctcaggt gatcctcccgcgtgagccgg cagactgtca tttctccatg ggcacctctg 12240 aatgttgagg cgggtgatgggtgggaggtt tagattgtgc tgcctgcagg ggctcccatc 12300 cccatgccgt ggatgcaggaggtgccgtct gggttcctgc aaccacattc aagccaatac 12360 acatttactg agcgcttgttgtgtacctca tcctgggagc tgtaggcagc agcccagtgt 12420 tccttagctc ctagaaattctaggtcccct ctacattctt tgcatgtagg caggatgacc 12480 tggacctgca ctatccagtacagtagctgc tcaccacatg tgactcttta aatttaaatt 12540 aattaaaatt aaactcaattcagttcctca gttgcattag ccacatttca agtactcagt 12600 agacgcatgt ggctggtggctgaggtatgg atggtgcaga cgtagaacct ttccatcatt 12660 gtagaaaatt ctatcagacagcattgctcc ggccacctgc caggtggtcc tccgggagtg 12720 ctggtgcgga gtgctggtgccgagtgctca gagtgggttc gggttcagtc cctgaaccca 12780 agcatcctct gcacccagatcctgcggatc tgcacgcggt acacgcccga gcaggacacc 12840 atgaccttct cggacgggctgaccctgaac cggacccaga tgcacaacgc tggcttcggc 12900 cccctcaccg acctggtctttgccttcgcc aaccagctgc tgcccctgga gatggatgat 12960 gcggagacgg ggctgctcagcgccatctgc ctcatctgcg gaggtgggca gggggcctgg 13020 gtctgggggc tgggctgggacgggggtgca gccctggagt ctcttccagg gagctctttc 13080 aggccacctc tgttaggtatctctagaggg cagggtctgg tctgcaacta cacagcaagg 13140 gggccatgtg gggcctggactcctgttccc gatttctggg caacacccct tctagggagg 13200 ttaagagtga gggtttgagggtcggaccaa ccagggtcac ctcctggccg atgcatgacc 13260 ctgagcaggt tgctgaacttctctgggcct ccgtttctgt acagtggggg cggtaacggt 13320 ctctagctca tgaagttgatgggaggatta cggtggtaac agatactgtg caggtgccca 13380 gagcgagctc cagtgcttgttagttgctat tttattgttg tgatttctgc catttcatct 13440 ggtttccaga ataacaggggggagtgggag cctgcctggg aaccctctcc ctgcttgagg 13500 atggcactgc ccatttggggtcccatccca ctaactgggc tcagggaggg tttggggcac 13560 cccctcaccc tcagctcccgttgctccctt ttaagggcct ctgtaccctg cggcagcaga 13620 gaccccatgc cctgccctgtgtggggaggc gcctgcgagc tgccctcctc catggcctgg 13680 gcaggcacgc cccccggtggccgaggctgg gggtgcagct gtgttcccag ctgctcaggg 13740 ggtggttctg cttcctcagaccgccaggac ctggagcagc cggaccgggt ggacatgctg 13800 caggagccgc tgctggaggcgctaaaggtc tacgtgcgga agcggaggcc cagccgcccc 13860 cacatgttcc ccaagatgctaatgaagatt actgacctgc gaagcatcag cgccaagggt 13920 gaggctcaca gacctggaggggtaccggcc cccgacacct ggcccaggcc cccacatcca 13980 agccagcacc ccatgtctttgtgccaggac aatacgacac ctgtccccat ctgtgtctag 14040 gctgaggtcc cctagtgactccactttgcc gaggtggccc gcctgtgtca cctttgtgtg 14100 gtagttcaga tcgtggctctggaaccagac acgtgggtgt gtgtccttgt gtgggtcact 14160 caacagctcc tagctacagtttcccttccg agggcgggga taacattcgt gtttacagag 14220 gggtcgggat gatccctagcacacagcaca ggggaaggaa gggcttggcg tctagcccag 14280 gccggcagtc tggccctggagccggagttc gggaccactt tgccccattg ccaccagcct 14340 ctggacctgg gggcttaagagagctggctc gtgtcaaaga actgaatccc aagaaagatg 14400 ctaatatcag cagtattgatcttcccacct cgagccaggc ttgctggggc tgggggtggg 14460 agggctggcc cagcgtgctgacctctgccc cctcctttcc tgcaggggct gagcgggtga 14520 tcacgctgaa gatggagatcccgggctcca tgccgcctct catccaggaa atgttggaga 14580 actcagaggg cctggacactctgagcggac agccgggggg tgggggggcg ggacggtggt 14640 ggccttctgc agtaaaaagtgccctgatgc caccattgcc gtaaaaacta atgcccaatt 14700 gtgataagga gctaccggggtacacacggg gactggttca aatggggcat cgccgaagca 14760 tgtgatgcta tgaacttaatcggactatta ttctggtgga tcctcaaacc agcatcgcaa 14820 cctggacact cttttgcatggtcgttatta tctccggtag actccttgcc tcccttttac 14880 ataaaaaggc ctcccccgacaaaaagggtc agttcgatcc ccactttcgg ttcgggagcc 14940 taccgtgtgc caaaggcccttaatctcgaa aatatcccaa ttacctgatg tcgtgcgacg 15000 cctaaaaatt ccccgtgttgccaccactgc ttgaaacccc caagcttggg tgttaatccc 15060 gaattggggg ccccccgtnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15120 nnnnnnnngg gcccccccgccaggcagctg tagccccagc ctcagcccca gctccaacag 15180 aagcagcccg gccacccactccccgtgacc gcccacgcca catggacaca gccctcgccc 15240 tccgccccgg cttttctctgcctttctacc gaccatgtga ccccgcacca gccctgcccc 15300 cacctgccct cccgggcagtactggggacc ttccctgggg gacggggagg gaggaggcag 15360 cgactccttg gacagaggcctgggccctca gtggactgcc tgctcccaca gcctgggctg 15420 acgtcagagg ccgaggccaggaactgagtg aggcccctgg tcctgggtct caggatgggt 15480 cctgggggcc tcgtgttcatcaagacaccc ctctgcccag ctcaccacat cttcatcacc 15540 agcaaacgcc aggacttggctcccccatcc tcagaactca caagccattg ctccccagct 15600 ggggaacctc aacctcccccctgcctcggt tggtgacaga gggggtggga caggggcggg 15660 gggttccccc tgtacataccctgccatacc aaccccaggt attaattctc gctggttttg 15720 tttttatttt aatttttttgttttgatttt tttaataaga attttcattt taagcacatt 15780 tatactgaag gaatttgtgctgtgtattgg ggggagctgg atccagagct ggagggggtg 15840 ggtccggggg agggagtggctcggaagggg cccccactct cctttcatgt ccctgtgccc 15900 cccagttctc ctcctcagccttttcctcct cagttttctc tttaaaactg tgaagtacta 15960 actttccaag gcctgccttcccctccctcc cactggagaa gccgccagcc cctttctccc 16020 tctgcctgac cactgggtgtggacggtgtg gggcagccct gaaaggacag gctcctggcc 16080 ttggcacttg cctgcacccaccatgaggca tggagcaggg cagagcaagg gccccgggac 16140 agagttttcc cagacctggctcctcggcag agctgcctcc cgtcagggcc cacatcatct 16200 aggctcccca gcccccactgtgaaggggct ggccaggggc ccgagctgcc cccacccccg 16260 gcctcagcca ccagcacccccatagggccc ccagacacca cacacatgcg cgtgcgcaca 16320 cacacaaaca cacacacactggacagtaga tgggccgaca cacacttggc ccgagttcct 16380 ccatttccct ggcctgccccccacccccaa cctgtcccac ccccgtgccc cctccttacc 16440 ccgcaggacg ggcctacaggggggtctccc ctcacccctg cacccccagc tgggggagct 16500 ggctctgccc cgacctccttcaccaggggt tggggcccct tcccctggag cccgtgggtg 16560 cacctgttac tgttgggctttccactgaga tctactggat aaagaataaa gttctattta 16620 ttctacacat gcctccagccttgctgcctc caccccctcc tcttggcgtc tggtctgggg 16680 gcttgggatg ggtttcgtcatgtgctctgg gcctgtgatg gccaggaatg agcactgggg 16740 ccaaggggct ggccagggcacccttccaag ctgccttctg aggcttacct tgtgctgggg 16800 tctttggaga tgctgagaaggagaaagtcc tgccccttgg gaagccctca gtctggggat 16860 ccacactgcc catgtcaaggagccccagtc tgggagtggg agagaagagg aggaaagctg 16920 cccccacctt cagggaacccccagtctgag ggaggaagcc ggagccaccc ctagacattt 16980 ctggtccttg ggaagccttc17000 4 459 PRT Rattus norvegicus 4 Met Tyr Glu Ser Val Glu Val Gly GlyLeu Thr Pro Ala Pro Asn Pro 1 5 10 15 Phe Leu Val Val Asp Phe Tyr AsnGln Asn Arg Ala Cys Leu Leu Gln 20 25 30 Glu Lys Gly Leu Pro Ala Pro GlyPro Tyr Ser Thr Pro Leu Arg Thr 35 40 45 Pro Leu Trp Asn Gly Ser Asn HisSer Ile Glu Thr Gln Ser Ser Ser 50 55 60 Ser Glu Glu Ile Val Pro Ser ProPro Ser Pro Pro Pro Leu Pro Arg 65 70 75 80 Ile Tyr Lys Pro Cys Phe ValCys Gln Asp Lys Ser Ser Gly Tyr His 85 90 95 Tyr Gly Val Ser Ala Cys GluGly Cys Lys Gly Phe Phe Arg Arg Ser 100 105 110 Ile Gln Lys Asn Met ValTyr Thr Cys His Arg Asp Lys Asn Cys Ile 115 120 125 Ile Asn Lys Val ThrArg Asn Arg Cys Gln Tyr Cys Arg Leu Gln Lys 130 135 140 Cys Phe Glu ValGly Met Ser Lys Glu Ser Val Arg Asn Asp Arg Asn 145 150 155 160 Lys LysLys Lys Glu Thr Pro Lys Pro Glu Cys Ser Glu Ser Tyr Thr 165 170 175 LeuThr Pro Glu Val Gly Glu Leu Ile Glu Lys Val Arg Lys Ala Asn 180 185 190Gln Glu Thr Phe Pro Ala Leu Cys Gln Leu Gly Lys Tyr Thr Thr Asn 195 200205 Asn Ser Ser Glu Gln Arg Val Ser Leu Asp Ile Asp Leu Trp Asp Lys 210215 220 Phe Ser Glu Leu Ser Thr Lys Cys Ile Ile Lys Thr Val Glu Phe Ala225 230 235 240 Lys Gln Leu Pro Gly Phe Thr Thr Leu Thr Ile Ala Asp GlnIle Thr 245 250 255 Leu Leu Lys Ala Ala Cys Leu Asp Ile Leu Ile Leu ArgIle Cys Thr 260 265 270 Arg Tyr Thr Pro Glu Gln Asp Thr Met Thr Phe SerAsp Gly Leu Thr 275 280 285 Leu Asn Arg Thr Gln Met His Asn Ala Gly PheGly Pro Leu Thr Asp 290 295 300 Leu Val Phe Ala Phe Ala Asn Gln Leu LeuPro Leu Glu Met Asp Asp 305 310 315 320 Ala Glu Thr Gly Leu Leu Ser AlaIle Cys Leu Ile Cys Gly Asp Arg 325 330 335 Gln Asp Leu Glu Gln Pro AspLys Val Asp Met Leu Gln Glu Pro Leu 340 345 350 Leu Glu Ala Leu Lys ValTyr Val Arg Lys Arg Arg Pro Ser Gln Pro 355 360 365 His Met Phe Pro LysMet Leu Met Lys Ile Thr Asp Leu Arg Ser Ile 370 375 380 Ser Ala Lys GlyAla Glu Arg Val Ile Thr Leu Lys Met Glu Ile Pro 385 390 395 400 Gly SerMet Pro Pro Leu Ile Gln Glu Met Leu Glu Asn Ser Glu Gly 405 410 415 LeuAsp Thr Leu Ser Gly Gln Ser Gly Gly Gly Thr Arg Asp Gly Gly 420 425 430Gly Leu Ala Pro Pro Pro Gly Ser Cys Ser Pro Ser Leu Ser Pro Ser 435 440445 Ser His Arg Ser Ser Pro Ala Thr Gln Ser Pro 450 455

What is claimed is:
 1. An isolated nucleic acid molecule consisting of anucleotide sequence selected from the group consisting of: (a) anucleotide sequence that encodes a polypeptide having an amino acidsequence comprising SEQ ID NO:2; (b) a nucleotide sequence consisting ofSEQ ID NO:1; (c) a nucleotide sequence consisting of SEQ ID NO:3; and(d) a nucleotide sequence that is completely complementary to anucleotide sequence of (a)-(c).
 2. A vector comprising the nucleic acidmolecule of claim
 1. 3. A host cell containing the vector of claim
 2. 4.A process for producing a polypeptide comprising culturing the host cellof claim 3 under conditions sufficient for the production of saidpolypeptide, and recovering said polypeptide.
 5. An isolatedpolynucleotide consisting of the nucleotide sequence of SEQ ID NO:1. 6.An isolated polynucleotide consisting of the nucleotide sequence of SEQID NO:3.
 7. The vector of claim 2, wherein said vector is selected fromthe group consisting of a plasmid, a virus, and a bacteriophage.
 8. Thevector of claim 2, wherein said isolated nucleic acid molecule isinserted into said vector in proper orientation and correct readingframe such that a polypeptide comprising SEQ ID NO:2 may be expressed bya cell transformed with said vector.
 9. The vector of claim 8, whereinsaid isolated nucleic acid molecule is operatively linked to a promotersequence.